USER GUIDE

For Research Use Only. Not for use in diagnostic procedures.

SuperScript® Choice System for cDNA Synthesis

Catalog number 18090-019

Document Part Number18090Publication Number MAN0000420Revision 3.0

Table of Contents

i

1. Notices to Customer .................................................................................1 1.1 Precautions ........................................................................................................1 1.2 Limited Label License No. 358: Research Use Only .......................................1 1.3 Trademarks ........................................................................................................1 1.4 Disclaimer...........................................................................................................1

2. Overview ........................................................................................................2 2.1 cDNA Libraries ...................................................................................................2 2.2 mRNA Isolation ..................................................................................................2 2.3 Choosing the Priming Method ..........................................................................4 2.4 First-Strand Synthesis .......................................................................................5 2.5 Second-Strand Synthesis .................................................................................6 2.6 Maximizing Ligation Efficiency by Adapter Addition .........................................6 2.7 Size Fractionation of cDNA ...............................................................................7 2.8 Choosing the Cloning Vector ............................................................................7 2.8.1 Plasmid Vectors .................................................................................... 8 2.8.2 λ Vectors................................................................................................ 8 2.9 Ligation of Size-Fractionated cDNA to the Vector of Choice .........................10

3. Methods ........................................................................................................11 3.1 Components ....................................................................................................11 3.2 General Comments .........................................................................................11 3.2.1 mRNA Purification................................................................................11 3.2.2 Advance Preparations .........................................................................12 3.2.3 Time Planning ......................................................................................12 3.2.4 Utilization of Reagents .........................................................................12 3.3 First-Strand Synthesis .....................................................................................14 3.4 Second-Strand Synthesis ...............................................................................16 3.5 EcoR I (Not I) Adapter Addition .......................................................................17 3.6 Phosphorylation of EcoR I-Adapted cDNA .....................................................17 3.7 Column Chromatography ................................................................................17 3.8 Ligation of cDNA to a Plasmid Vector .............................................................21 3.9 Ligation of cDNA to λgt11 and λgt10 Vectors ................................................21 3.10 Ligation of cDNA to a λZipLox Vector .............................................................22 3.11 Analysis of cDNA Products .............................................................................22 3.11.1 First-Strand Yield ................................................................................22 3.11.2 Second-Strand Yield ...........................................................................23 3.11.3 Gel Analysis ........................................................................................23 3.12 Analysis of cDNA from the cDNA Size Fractionation Column .......................24

4. Troubleshooting .......................................................................................26 4.1 Isolation of mRNA ............................................................................................26 4.2 First-Strand Reaction ......................................................................................26 4.3 Second-Strand Reaction .................................................................................27 4.4 EcoR I (Not I) Adapter Addition .......................................................................27 4.5 Phosphorylation of EcoR I-Adapted cDNA .....................................................27

ii

4.6 Column Chromatography ................................................................................28 4.7 Ligation of cDNA to a Plasmid Vector .............................................................28 4.8 Ligation of cDNA to a λ Vector ........................................................................28

5. References ..................................................................................................29

6. Related Products ......................................................................................30

Figures1. Summary of the SuperScript® Choice System Procedure ........................................32. Effect of Random Hexamer Concentration on First-Strand cDNA Synthesis ...........43. Sequence of the EcoR I (Not I) Adapter .....................................................................74. Detailed Protocol Flow Diagram ...............................................................................135. Alkaline Agarose Gel Analysis of First and Second-Strand cDNA Synthesized with the SuperScript® Choice System. .....................................246. Electrophoretic Analysis of Size-Fractionated cDNA ...............................................25

Table of Contents

1Notices to Customer

1

1.1 PrecautionsWarning: This product contains hazardous reagents. Consult the applicable SDS(s) before using this product. Disposal of waste organics, acids, bases, and radioactive materials must comply with all appropriate federal, state, and local regulations.

1.2 Limited Label License No. 358: Research Use OnlyThe purchase of this product conveys to the purchaser the limited, non-transferable right to use the purchased amount of the product only to perform internal research for the sole benefit of the purchaser. No right to resell this product or any of its components is conveyed expressly, by implication, or by estoppel. This product is for internal research purposes only and is not for use in commercial applications of any kind, including, without limitation, quality control and commercial services such as reporting the results of purchaser's activities for a fee or other form of consideration. For information on obtaining additional rights, please contact outlicensing@lifetech.com or Out Licensing, Life Technologies, 5791 Van Allen Way, Carlsbad, California 92008.

1.3 TrademarksThe trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners.

Sephacryl is a registered trademark of GE Healthcare Bio-Sciences.

TRIzol is a registered trademark of Molecular Research Center, Inc.

RNase Away and ART are registered trademarks of Molecular Bio-Products, Inc.

1.4 DisclaimerLIFE TECHNOLOGIES CORPORATION AND/OR ITS AFFILIATE(S) DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE, OR NON-INFRINGEMENT. TO THE EXTENT ALLOWED BY LAW, IN NO EVENT SHALL LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF.

©2013 Life Technologies Corporation. All rights reserved.

2

2

Overview

2.1 cDNA LibrariesA cDNA library is an array of DNA copies of an mRNA population that are propagated in a cloning vector and usually maintained in E. coli. A good cDNA library is large enough to contain representatives of all sequences of interest, some of which may be derived from low-abundance mRNAs. Depending on the research objective, cDNA library construction can begin by priming first-strand synthesis with oligo(dT) or with random hexamers. These two priming methods will ultimately produce two different types of cDNA libraries, each of which will serve a different purpose. Libraries produced using oligo(dT) priming will contain cDNA inserts of the largest size; libraries produced using random hexamer priming may contain a higher proportion of cDNA with the 5´-most sequence information and may include cDNA copies of poly(A)- as well as poly(A)+ RNA. Careful planning and appropriate choices will result in cDNA libraries tailored to specific research objectives.

Because a cDNA library is the end product of many individual steps, its quality can be compromised by inefficiency at any point in the procedure. The SuperScript® Choice System integrates state-of-the-art cDNA synthesis with simplified downstream technology to produce cDNA that can be ligated to any EcoR I-digested vector for subsequent introduction into E. coli. If the starting mRNA is of high quality, the cDNA library constructed with this system will satisfy the preceding criteria.

cDNA libraries can be broadly classified as directional or random. Whereas all members of a directional library contain cDNA inserts cloned in a specific orientation relative to the transcriptional polarity of the original mRNAs, members of random libraries contain cDNA inserts cloned in either orientation. For maximum versatility, the SuperScript® Choice System has been designed for generation of random libraries: the double-stranded, EcoR I-ended cDNAs produced with this system are suitable for insertion into the vast majority of existing vectors.

The major steps in constructing a random cDNA library from an mRNA population using the SuperScript® Choice System are summarized in Figure 1.

2.2 mRNA IsolationConstruction of a good cDNA library begins with the preparation of high-quality mRNA. The quality of the mRNA dictates the maximum amount of sequence information that can be converted into cDNA. Thus, it is important to optimize the isolation of mRNA from a given biological source and to prevent adventitious introduction of RNases into a preparation that has been carefully rendered RNase-free. For optimal results, the mRNA must be purified over an affinity column [oligo(dT) cellulose being the most commonly used matrix] to select the polyadenylated [poly(A)+] RNA (1). Since the vast majority of mRNA is poly(A)+, this selection operationally defines the mRNA population. Typically, 0.5% to 2% of a total RNA population is mRNA, so isolation of this fraction from rRNA, tRNA, and degraded mRNA enhances the synthesis of first-strand cDNA and minimizes spurious transcription of non-mRNAs. An mRNA preparation that has undergone two selections over this matrix (in which the eluate from one round of purification has been bound to the column and eluted a second time) will produce the highest quality mRNA. When properly prepared, oligo(dT) cellulose-purified RNA will be ≥90% mRNA.

2

3

The amount of mRNA needed to prepare a library is dependent on the efficiency of the individual steps needed to convert the mRNA into a form that can be cloned and on the efficiency with which the recombinant molecules can be introduced into a host. Generally, 1 to 5 µg of mRNA will be sufficient to construct a cDNA library containing 106 to 107 clones in E. coli.

Figure 1. Summary of the SuperScript® Choice System procedure.

Overview 2.3 Choosing the Priming MethodFirst-strand cDNA synthesis is most commonly primed using oligo(dT) or modifications of this sequence (such as primer-adapters) that bind to the poly(A) tail of mRNA. This priming method offers two major advantages: only poly(A)+ RNA is copied, and most cDNA clones begin at the 3´ terminus of the mRNA. At the same time, oligo(dT) priming has certain limitations: some cDNA clones may not be full length, due to RNA secondary structure or pausing by reverse transcriptase, and poly(A)- mRNA cannot be copied.

An alternative method is to use random hexamers, which, in theory, are capable of binding and priming throughout virtually any RNA template. Random hexamers, which may be used either by themselves or in combination with oligo(dT), have been instrumental in producing cDNAs containing more 5´ information than those primed with oligo(dT) alone (2,3). In addition, random hexamers can be used to generate cDNA libraries from poly(A)- mRNA (4) and single-stranded viral RNAs (2,5).

Although increasing the random hexamer concentration increases the percentage yield of first-strand cDNA synthesis, it also decreases the average cDNA size. A typical random hexamer titration profile (using cDNA synthesized from HeLa mRNA), as compared to the result obtained with oligo(dT) priming, is shown in figure 2. Identical random hexamer titration profiles are obtained whether the first-strand cDNA reaction is incubated at 37°C or 45°C, using from 1–5 µg of mRNA per reaction, and in the presence or absence of oligo(dT) primers. Note: A certain amount of self-priming by subpopulations of mRNA may occur in the absence of any exogenous primers (see lane 2 of figure 2), which can contribute nominally (~6%) to the overall first-strand cDNA yield produced by random hexamer priming.

4

Figure 2. Effect of random hexamer concentration on first-strand cDNA synthesis. 2 µg of HeLa mRNA was primed with 1 µg of oligo(dT)12-18 (lane 1) or various amounts of random hexamers (lanes 2 through 9).

1 2 3 4 5 6 7 8 9

Kb

12.2—

7.1—5.1—

4.0—

3.0—

2.0—

1.6—

1.0—

0.5—

| | | | | | | | | 1 µg 0 5 20 60 150 400 1,000 5,000

5

The SuperScript® Choice System includes both oligo(dT)12-18 and random hexamer primers. In the decision to use either priming method separately, or both in combination, to synthesize cDNA, the following considerations should be noted:1. Priming with oligo(dT) by itself is decidedly better at producing larger cDNA

inserts.2. Random hexamer priming yields cDNA that are smaller on average but that

may better represent the entire RNA template. Priming with random hexamers at a concentration of 50–150 ng per reaction generally yields twice the amount of 5´-end information of the β-actin mRNA as oligo(dT) priming (6). For some RNAs, however, higher concentrations of random hexamers may be needed to increase the proportion of cDNA containing the 5´-most sequence information.

3. Random hexamers and oligo(dT), when used in combination, should be added simultaneously to ensure all possible priming events.

2.4 First-Strand SynthesisAvian myeloblastosis viral (AMV) reverse transcriptase (RT) was the first enzyme used to synthesize cDNA in vitro, and much of the early work in cDNA synthesis and cloning was developed using this enzyme. However, the successful cloning of the Moloney murine leukemia virus (M-MLV) RT (7) has provided researchers with an alternative enzyme. The cloned M-MLV RT gene has been further engineered to produce a novel enzyme (SuperScript® II RT) with reduced RNase H activity (8). This modification is significant because RNase H activity is detrimental to the first-strand cDNA synthesis reaction in two ways:1. The initiation of first-strand synthesis depends upon the hybridization of a

primer to the mRNA, usually at the poly(A) tail. This hybrid is a substrate, not only for the polymerase activity of the RT but also for the RNase H activity (9). In the resulting competition between these two activities, the extent to which the RNase H activity destroys the hybrid prior to the initiation of polymerization determines the maximal number of initiation events that can actually occur. Hydrolysis of the RNA in the hybrid reduces the maximal yield of cDNA by effectively removing a portion of the mRNA from the reaction.

2. When the RT is synthesizing the first-strand cDNA, the RNase H activity will quickly degrade the template that has already been copied because the mRNA is in hybrid form as a result of the polymerization reaction. If the scissions in the mRNA occur near the point of chain growth, the uncopied portion of the mRNA can dissociate from the transcriptional complex, resulting in termination of cDNA synthesis for that template and consequent reduction in the yield of full-length cDNA. This problem can be exacerbated if the RT pauses during transcription at certain primary or secondary structural domains.

When used with synthetic RNA produced in vitro, SuperScript® II RT has demonstrated significantly greater full-length cDNA synthesis and higher yields of first-strand cDNA than other commercially available RTs (10, 11, 12).

The reaction conditions for first-strand synthesis catalyzed by SuperScript® II RT have been optimized for yield and size of the cDNAs. The optimal first-strand reaction temperature for SuperScript® II RT is 37°C; however, should secondary structure make reverse transcription difficult, a higher reaction temperature may be used. SuperScript® II RT is stable at 45° to 50°C and can be used at this temperature, if necessary.

The amount of mRNA can be as high as 5 µg in a 20-µL first-strand cDNA synthesis reaction. We recommend using at least 1 µg of mRNA so that there will be sufficient material at the end of the procedure to obtain the required number of clones. The amount of SuperScript® II RT needed in the first-strand reaction varies linearly with the amount of mRNA: 200 units of SuperScript® II RT for ≤1 µg of mRNA, and 200 units per µg for 1–5 µg of mRNA. Although the exact ratio of SuperScript® II RT to mRNA is not critical, these approximate proportions have produced reliable results.

2

Overview 2.5 Second-Strand SynthesisThe primary sequence of the mRNA is recreated as second-strand DNA using the first-strand cDNA as a template. The SuperScript® Choice System uses nick translational replacement of the mRNA to synthesize the second-strand cDNA. First described by Okayama and Berg (13), and later popularized by Gubler and Hoffman (14), second-strand synthesis is catalyzed by E. coli DNA polymerase I in combination with E. coli RNase H and E. coli DNA ligase. Although RNase H is not essential if the first-strand synthesis is catalyzed by AMV or M-MLV RT, E. coli RNase H must be included in the second-strand reaction when SuperScript® II RT has been used for first-strand cDNA synthesis. E. coli DNA ligase has been shown to improve the cloning of double-stranded (ds) cDNA synthesized from longer (≥2 kb) mRNAs (15).

The first and second-strand syntheses are performed in the same tube without intermediate organic extraction or ethanol precipitation. This one-tube format speeds the synthesis procedure and maximizes recovery of cDNA. The efficiency of the second-strand reaction is influenced by the amount and concentration of the reactants, so the instructions must be followed as described for best results. The second-strand reaction is incubated at 16°C to prevent spurious synthesis by DNA polymerase I due to its tendency to strand-displace (rather than nick translate) at higher temperatures. The last step in the cDNA synthesis procedure is to ensure that the termini of the cDNA are blunt. This is easily done by adding T4 DNA polymerase to the second-strand reaction mixture and incubating briefly at 16°C. The cDNA is then deproteinized by organic extraction and precipitated with ethanol to render it ready for downstream manipulation.

2.6 Maximizing Ligation Efficiency by Adapter AdditionThe product of the first and second-strand synthesis reactions performed using the SuperScript® Choice System is blunt-ended cDNA, a poor substrate for T4 DNA ligase. To maximize ligation efficiency into the vector, the blunt ends of the cDNA are converted to termini that contain 5´ extensions by adding adapters to the cDNA. Adapters are short, duplex oligomers, blunt-ended at one terminus and containing a 4-base, 5´ extension at the other terminus. The blunt-end ligation of the adapters to the cDNA can be driven by adapter excess, much the way molecular linkers are added to DNA. However, unlike linkers, adapters contain preformed extensions and do not require restriction digestion to expose the termini.

The 4-base, 5´ extension of the adapters provided with the SuperScript® Choice System corresponds to the termini produced by digestion with EcoR I. The sequence of the EcoR I (Not I) adapter included in the SuperScript® Choice System is shown in Figure 3. Several details should be noted:1. The recognition sequences for Not I and Sal I are contained within the EcoR I

(Not I) adapter to allow easy release of cDNA inserts. Both restriction endonuclease sites are extremely rare in mammalian genomes, occurring approximately once in 106 bp (Not I) and 105 bp (Sal I).

2. Only one of the oligomers of the EcoR I (Not I) adapter is phosphorylated, which drives adapter-to-cDNA ligation and essentially eliminates cDNA-to-cDNA ligation.

6

2

7

Adding the EcoR I (Not I) adapters to the cDNA places the same EcoR I 5´ extension at both ends of the cDNA. The cDNA is then used to construct a random library by ligating it to an EcoR I-digested vector that has been dephosphorylated to reduce the background arising from self-ligation of the vector. This, in turn, requires phosphorylation of the adapted cDNA at its 5´ termini so that it can be ligated to the 5´-dephosphorylated, EcoR I-digested vector. The ligation and phosphorylation steps used in the SuperScript® Choice System are performed in the same buffer without any organic extraction or ethanol precipitation, which maximizes efficiency and facilitates cDNA recovery.

2.7 Size Fractionation of cDNASize fractionation of cDNA, following adapter addition and phosphorylation, is important because residual adapters are present in large molar excess and can impede vector ligation to cDNA by ligating to the EcoR I termini of the predigested vector. Size fractionation also reduces the tendency of smaller (<500 bp) inserts to predominate the library. These smaller cDNAs can arise for several reasons:1. Most mRNA preparations are not size-selected, so partially degraded mRNAs

can be selected on the oligo(dT) cellulose columns along with longer mRNAs. These will be reverse transcribed into small cDNAs.

2. If extreme care is not taken to prevent RNase contamination during first-strand synthesis, degradation can occur when the mRNA is manipulated.

3. Some mRNAs contain regions that are not readily reverse transcribed, and RT is not able to synthesize complete first strands.

Column chromatography is the simplest method of producing size-fractionated cDNA, free of adapters and other low molecular weight DNAs. The SuperScript® Choice System contains three 1-mL, disposable columns, prepacked with Sephacryl® S-500 HR, that quickly and easily remove DNAs <500 bp and size-fractionate cDNAs >500 bp, as well as perform an exchange of buffers, thus facilitating construction of libraries from fractions enriched for larger cDNA. The column chromatography buffer (described in Chapter 3) is formulated to allow the cDNA in the column factions to be ligated directly into predigested plasmid cloning vectors. Although the final yield of size-fractionated cDNA will depend upon the recovery at each step of the procedure, the average overall yield should be 5%–10% of the mass of the starting mRNA.

2.8 Choosing the Cloning VectorTwo types of vectors are generally used for cDNA cloning in E. coli plasmids and bacteriophage lambda (λ) derivatives. Several factors affect the choice of vector.

Ease of use. Cloning into plasmids is generally easier for the novice because it requires less manipulation of the cDNA and avoids potential problems when propagating phage. However, this consideration alone should not preclude using a λ vector if it is otherwise the best choice. For detailed discussions of cDNA cloning into λ vectors, see references 16 and 17.

5´-AATTCGCGGCCGCGTCGAC-3´3´- GCGCCGGCGCAGCTGp-5´

EcoR I Not I Sal I

Figure 3. Sequence of the EcoR I (Not I) adapter.

Overview Antibody screening. λ vectors are the better choice if the cDNA library is to be screened with the antibody (18). Although plasmid-based cDNA libraries may also be screened by this method (19), the process is more cumbersome and considerably more tedious.

Nucleic acid screening. Choosing between plasmid and λ vectors is less critical if the library will be screened with a nucleic acid probe because performance is similar in both systems (20,21). Other considerations (such as subcloning capability) may dictate which system to use.

The SuperScript® Choice System is designed to produce cDNA containing EcoR I-cohesive ends suitable for ligation to any EcoR l-digested, dephosphorylated plasmid or λ vector (consult reference 17 for details on preparing dephosphorylated vectors).

2.8.1 Plasmid VectorsThere are many plasmid vectors available that are compatible with the SuperScript® Choice System for cDNA Synthesis. Since the SuperScript® Choice System is designed to produce cDNA containing EcoR I-cohesive ends, the plasmid must contain a unique EcoR I site within the multiple cloning site. When choosing a plasmid consider what screening method will be used and if the library will be used in a subtractive cDNA library construction. If the library will be used for making RNA, in vitro translation, or subtraction procedures the multiple cloning site must be flanked by RNA polymerase promoters (i.e. SP6, T7, or T3 RNA polymerase promoters). A vector with an f1 origin of replication can be infected with M13K07 helper phage to produce single-strand plasmid DNA for sequencing or in vitro mutagenesis (22). Nested deletions for sequencing are facilitated by having restriction endonuclease sites in the multiple cloning site clustered together.

Plasmid shuttle vectors for transient expression of cloned genes in mammalian cells such as COS cells and DNA cloning in E. coli can also be used with the SuperScript® Choice System for cDNA Synthesis.

Plasmid pcDNA3 (+) is a multifunctional vector for cDNA cloning, in vitro transcription, and dideoxy sequencing. The plasmid contains a unique multiple cloning site with restriction sites for EcoR I and 11 other restriction endonucleases. There is a T7 RNA polymerase promoter that can be used to generate RNA for probes, in vitro translation, or subtracted cDNA libraries. The plasmid contains the CMV promoter, for expression of cloned genes. DNA inserts can be sequenced from double-stranded DNA using T7 forward or BGH reverse sequencing primers. Single-stranded plasmid DNA for sequencing or in vitro mutagenesis can be generated by infection of transformed cells with an appropriate helper phage such as M13K07 (22). The β-lactamase gene on the plasmid provides for convenient selection by ampicillin resistance.

2.8.2 λVectorsλgt10,EcoR I Arms is a precut version of λgt10, purified from bacteriophage DNA (imm434 b527), which has been prepared by ligation at the cos sites, digestion with EcoR I, and dephosphorylation. This vector contains an EcoR I site within the repressor gene and can accommodate inserts up to 7 kb in length (16). Interruption of the repressor gene by insertion of cDNA into the EcoR I site converts the phage from cl+ to cl-, which changes plaque morphology from turbid to clear when plated on an E. coli strain such as C600 (24). By using the bacterial strain C600hflA150 that contains a high frequency lysogeny mutation (16), lytic growth is repressed so effectively that plaque formation by cl+ phage does not occur; only cl- (recombinant) phage produce plaques, significantly reducing the background. Because λgt10 is not an expression vector, recombinant libraries prepared in λgt10 cannot be screened with antibodies.

8

2

9

λgt11,EcoR I Arms is a precut version of λgt11, purified from bacteriophage DNA (lac5 cl857 nin5 Sam100), which has been prepared by ligation at the cos sites, digestion with EcoR I, and dephosphorylation. Unlike λgt10, λgt11 is an expression vector: libraries prepared in λgt11 can be screened immunologically using specific antibodies (18,25), as well as with nucleic acid probes. This vector contains a unique EcoR I cloning site near the end of a β-galactosidase coding sequence and can accept inserts up to 7.2 kb in length. If cDNA containing an open reading frame is inserted into this site in the correct orientation, a fusion protein is produced when expression from the lac promoter is induced with isopropylthio-β-galactoside (IPTG). Thus, expression of proteins, which may be toxic to the cell, can be delayed until the library has been amplified and is ready for immunological screening. The lac promoter is also used to control expression of the cloned gene if large-scale synthesis is needed for purification. Insertion of cDNA into the lacZ gene of λgt11 will produce plaques that are usually colorless instead of blue on plates containing IPTG and X-gal. The ratio of colorless to blue plaques is often used to estimate the percentage of recombinants in the library.

λZipLox,EcoR I Arms is a precut version of λZipLox, which has been prepared by ligation at the cos sites, digestion with EcoR I, and dephosphorylation. Libraries prepared in λZipLox can be screened immunologically using specific antibodies (18,25), as well as with nucleic acid probes. λZipLox contains the plasmid pZL1, flanked by loxP sequences, between portions of the left and right arms of λgt10 and λgt11. cDNA inserts cloned into the EcoR I site of λZipLox reside within the inducible lac Z´ gene commonly found in pUC-type plasmid vectors. When the lac promoter is induced with IPTG, the cloned gene is expressed as a fusion protein embedded within the amino-terminal portion of the β-galactosidase fragment encoded by lac Z´. Following cloning and selection of desired clones, the cDNA can be recovered in the autonomously replicating multifunctional plasmid pZL1 using a simple in vivo excision protocol, obviating the need for tedious subcloning.

Life Technologies vector sequences, restriction information, and maps can be found in the Vector Data area of our web site www.lifetechnologies.com.

2.9 Ligation of Size-Fractionated cDNA to the Vector of ChoiceThe ligation reactions described in Sections 3.8, 3.9 and 3.10 in Chapter 3 will suffice for most applications. We have found that 10–20 ng of cDNA saturates 50 ng of plasmid vector and that 50 ng of cDNA saturates 500 ng of λ vector; to use more cDNA is wasteful (as little as 1 ng of cDNA can be used in either ligation reaction). Foranyparticularpopulationof cDNA,however, these ratiosmaynot be optimal. If the described ligation conditions do not yield enough transformantsorplaquestomakeyourlibrarycomplete,thevector-to-cDNAratio yielding the maximum number of clones should be determined empirically.The yield is also dependent upon the transformation efficiency of the E. coli cells used to plate the plasmid-based library or the efficiency of the in vitro packaging methods used to introduce the λ-ligated cDNA into E. coli by infection. For either type of vector, the following considerations should be noted: Plasmid Vectors. If the E. coli competent cells yield ~1 × 109 transformants/µg

of pUC19 plasmid DNA, then the ligated cDNA should yield 0.5 to 1 × 107 transformants/µg of vector; this is equivalent to 2.5 to 5 × 107 transformants/µg of cDNA. Thus, a plasmid library containing 5 × 105 clones can be constructed from 10 ng of cDNA used in the ligation reaction in Section 3.8. Plasmid-ligated cDNA can also be introduced into E. coli cells by electroporation, which generally will yield a greater number of transformants (2.5 × 108 to 1 × 109 transformants/µg cDNA) from the same amount of ligated cDNA. Electroporation may be especially useful if the library must be very large or if you have <10 ng of cDNA.

λVectors. If the in vitro packaging extract yields approximately 5 × 109 plaque forming units (pfu)/µg of ligated, wild-type λ DNA, then the ligated cDNA should yield 0.5 × 107 to 1 × 107 pfu/µg of vector; this is equivalent to 2.5 × 108 to 5 × 108 pfu/µg of cDNA. Thus, a library containing 2 × 106 clones can be constructed from as little as 10 ng of cDNA.

Overview

10

3

11

3.1 ComponentsThe components of the SuperScript® Choice System for cDNA Synthesis are as follows. Components are provided in sufficient quantities to perform three separate experiments, each converting up to 5 µg of mRNA into size-fractionated, EcoR I-adapted cDNA, ready for ligation into any EcoR I-digested, dephosphorylated vector. Store chromatography columns (Part No. 8092CL) at 2°C to 8°C and store the reagent assembly (Part No. 8090RT) at –30°C to –10°C.

Component AmountOligo(dT)12-18 primer (0.5 µg/µL) .......................................................................................... 10 µLRandom Hexamers (50 ng/µL) ........................................................................................... 50 µL 5X First-Strand Buffer [250 mM Tris-HCl (pH 8.3),

375 mM KCl, 15 mM MgCl2] .......................................................................................... 1 mL0.1 M DTT ......................................................................................................................... 250 µL10 mM dNTP Mix (10 mM each dATP, dCTP, dGTP, dTTP) ............................................... 20 µLSuperScript® II RT (200 units/µL) ....................................................................................... 50 µL 5X Second-Strand Buffer [100 mM Tris-HCl (pH 6.9),

450 mM KCl, 23 mM MgCl2, 0.75 mM ß-NAD+, 50 mM (NH4)2SO4] ........................... 500 µLE. coli DNA Ligase (10 units/µL) ......................................................................................... 10 µLE. coli DNA Polymerase I (10 units/µL) .............................................................................. 50 µLE. coli RNase H (2 units/µL) ............................................................................................... 20 µLT4 DNA Polymerase (5 units/µL) ........................................................................................ 10 µL5X Adapter Buffer [330 mM Tris-HCl (pH 7.6), 50 mM MgCl2, 5 mM ATP] ............................................................................................ 30 µLEcoR I (Not I) Adapters (1 µg/µL) ....................................................................................... 30 µLT4 DNA Ligase (1 units/µL) ................................................................................................ 50 µLT4 Polynucleotide Kinase (10 units/µL) .............................................................................. 10 µL5X T4 DNA Ligase Buffer [250 mM Tris-HCl (pH 7.6),

50 mM MgCl2, 5 mM ATP, 5 mM DTT, 25% (w/v) PEG 8000] ....................................... 1 mLDEPC-treated Water ....................................................................................................... 1.25 mLControl RNA (0.5 µg/µL) ..................................................................................................... 15 µLYeast tRNA (1 µg/µL) ........................................................................................................ 100 µLcDNA size fractionation columns ......................................................................................... threeManual ................................................................................................................................... one

3.2 General Comments3.2.1 mRNA PurificationOne of the most important steps preceding the synthesis of cDNA and the establishment of a library is isolation of intact mRNA. The Micro-FastTrack® 2.0 mRNA Isolation Kit allows you to isolate mRNA in a fast and convenient procedure.

Successful cDNA synthesis demands an RNase-free environment at all times, which will generally require the same level of care used to maintain aseptic condi tions when working with microorganisms. Several additional guidelines should be followed:1. Never assume that anything is RNase-free, except sterile pipets, centrifuge

tubes, culture tubes, and any similar labware that is explicitly stated to be sterile.

2. Dedicate a separate set of automatic pipets for manipulating RNA and the

Methods

Methods buffers and enzymes used to synthesize cDNA. All purpose pipets may be quickly prepared for RNA use by wiping the outside surface of the pipet with RNase AWAY® Reagent. Use barrier tips such as ART® Tips.

3. Obtain RNase-free microcentrifuge tubes or treat tubes overnight in a 0.01% (v/v) aqueous solution of diethylpyrocarbonate (DEPC); rinse them with autoclaved, distilled water; and autoclave them.

4. Avoid using any recycled glassware unless it has been specifically rendered RNase-free by rinsing with 0.5 N NaOH or RNase AWAY® Reagent followed by copious amounts of DEPC-treated, autoclaved water or other prepared RNase-free water. Alternatively, bake glassware at 150°C for 4 hours.

5. Use reagent grade solutions that are set aside for RNA use only. The preferred method of obtaining RNase-free solutions is to treat them with 0.01% (v/v) DEPC followed by autoclaving. If preparing solutions containing primary amines (such as Tris), where DEPC cannot be used, or preparing heat-sensitive solutions, use RNase-free reagents, water and labware, and filter the solution through a 0.2-µm disposable, sterile filter.

3.2.2 Advance PreparationsBefore using this system, please review the protocol flow diagram in figure 4. You will need the following items not included in this system:• autoclaved 1.5-mL microcentrifuge tubes• microcentrifuge capable of generating a relative centrifugal force of 14,000 × g• automatic pipets capable of dispensing 1 to 20 µL, 20 to 200 µL, and 200 µL

to 1 mL• autoclaved, disposable tips for automatic pipets• disposable gloves• 16°C and 37°C water baths• 1–10 µCi [α-32P]dCTP (400 to 3,000 Ci/mmol)• 500 mL 10% (w/v) TCA (trichloroacetic acid) containing 1% (w/v) sodium

pyrophosphate (store at 4°C)• glass fiber filters (1 × 2 cm) (Whatman GF/C or equivalent)• buffer-saturated phenol:CHCl3:isoamyl alcohol [25:24:1 (v/v/v)]• TEN buffer [10 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 25 mM NaCl; autoclaved]• 7.5 M ammonium acetate (NH4OAc) filtered through a sterile, 0.2-µm

nitrocellulose filter• 70% (v/v) ethanol (–20°C)

3.2.3 Time PlanningStarting with poly(A)+ RNA, these protocols are designed to yield cDNA containing EcoR I-cohesive ends in ~2 days. For best results, the procedure should be completed as quickly as possible because radiochemical effects induced by the decay of the 32P in the cDNA can diminish transformation efficiencies over time.We recommend that the protocols be completed as follows:

Day 1: Sections 3.3 and 3.4, and steps 1 and 2 of Section 3.5 (overnight incubation of EcoR I (Not I) Adapter Addition reaction).

Day 2: Sections 3.5 through 3.8 or 3.9.If interrupting the procedure at any other point becomes necessary, you may do so following Sections 3.4 or 3.7. When stopping at any such point, always store the cDNA as the uncentrifuged ethanol precipitate at –20°C to minimize the aforementioned effects of 32P decay.

3.2.4 Utilization of ReagentsReagents included with the SuperScript® Choice System are provided in sufficient quantities to perform three complete experiments converting up to 5 µg of mRNA into size-fractionated, EcoR I-adapted cDNA. Additionally, the components for first

12

Note: Suggested stopping points are noted in the protocol with the

icon.

Note: The use of pre-made phenol: chloroform:isoamyl alcohol (25:24:1, v/v) is recommended. If making your own, saturate the redistilled phenol with TEN buffer, not with distilled water.

3

13

Figure 4. Detailed protocol flow diagram.

Poly(A)+ mRNA

Oligo(dT)12-18, Random Hexamers,or bothDEPC-Treated Water

5X First-Strand Buffer40.1 M DTT10 mM dNTP Mix[α-32P]dCTP

SuperScript® II RT

First-strand reaction

DEPC-treated water5X Second-Strand Buffer10 mM dNTP MixE. coli DNA LigaseE. coli DNA Polymerase IE. coli RNase H

Transfer to ice

First-strand cDNA

T4 DNA Polymerase

Extract, precipitate

Second-strand reaction

70°C, 10 min

37°C, 2 min

37°C, 1 h

16°C, 2 h

16°C, 5 min

DEPC-Treated Water5X Adapter BufferEcoR I (Not I) Adapters0.1 M DTTT4 DNA Ligase

ds cDNA

T4 Polynucleotide Kinase

Column chromatography

EcoR I-adapted cDNA

37°C, 30 min70°C, 10 min

16°C, 16 h70°C, 10 min

Size-fractionated cDNA

Remove aliquot for yield and gel electrophoretic analyses

Methods and second-strand synthesis are provided in sufficient quantities to perform Sections 3.3 and 3.4 five times. You may wish to use these extra quantities to test a small amount of your mRNA by determining first or second-strand yield and visualizing the distribution of the products by gel electrophoresis. Alternatively, the extra components can be held as a backup in case of accidental loss of material or procedural error.

3.3 First-Strand SynthesisThe 20-µL reaction described is designed to convert up to 5 µg of mRNA into first-strand cDNA. The amount of SuperScript® II RT added to the reaction will be dependent upon the amount of starting mRNA. We recommend 200 units of SuperScript® II RT for ≤1 µg of mRNA and 200 units/µg of mRNA for 1–5 µg of mRNA.

If second-strand cDNA is to be labeled instead of first-strand cDNA, the first-strand reaction should be set up without [α-32P]dCTP (adjust the amount of water in the reaction to maintain the 20-µL final volume), and the reaction should be carried through the second-strand synthesis procedure as described in Section 3.4. In this case, add 1 µL (10 µCi/µL) [α-32P]dCTP to the second-strand reaction after the 10 mM dNTP mix is added.

A single-transcript control RNA is included in the SuperScript® Choice System as an aid in verifying the first-strand reaction. If you decide to use the control RNA, simply substitute 4 µL (2 µg) in the first-strand reaction for your mRNA.

1. Perform one of the following substeps – a, b, or c – depending on your choice of priming method.

a. For priming with oligo(dT): Add 2 µL of Oligo(dT)12-18 primer to a sterile 1.5-mL microcentrifuge tube. Add mRNA, diluted as needed with DEPC-treated water, according to the following table:

µg of mRNA ≤1 2 3 4 5

mRNA (plus DEPC-treated water) 9 8 7 6 5 to a total volume (µL) 11 10 9 8 7

b. For priming with random hexamers: Add 1 to 3 µL (50 to 150 ng) of Random Hexamers to a sterile 1.5-mL microcentrifuge tube. Add mRNA, diluted as needed with DEPC-treated water, according to the following table:

µg of mRNA ≤1 2 3 4 5

mRNA (plus DEPC-treated water) to a total volume (µL) 11 10 9 8 7

c. For priming with both Oligo(dT) and Random Hexamers: Add 2 µL of Oligo(dT)12-18 Primer and 1 to 3 µL (50 to 150 ng) of Random Hexamers to a sterile 1.5-mL microcentrifuge tube. Add mRNA, diluted as needed with DEPC-treated water, according to the following table:

µg of mRNA ≤1 2 3 4 5

mRNA (plus DEPC-treated water) to a total volume (µL) 11 10 9 8 7

14

Note: The efficiency of the second-strand reaction is influenced by the amount and concentration of the reactants, so the instructions must be followed as described for best results.

Note: Do not proceed with this protocol until you have made the appropriate decisions regarding your choice of primer. For more information, see Section 2.3, Choosing the Priming Method.

3

15

2. Heat the mixture to 70°C for 10 minutes and quick-chill on ice. Collect the contents of the tube by brief centrifugation and add the following:

Component Volume (µL) 5X First-Strand Buffer 4 0.1 M DTT 2 10 mM dNTP Mix 1 [α-32P]dCTP (1 µCi/µL) 1

The total volume should now correspond to the following table: µg of mRNA (from step 1)

≤1 2 3 4 5

Total volume (µL) 19 18 17 16 15

3. Mix the contents of the tube by gently vortexing and collect the reaction by brief centrifugation. Place the tube at 37°C for 2 minutes to equilibrate the temperature.

4. Add SuperScript® II RT according to the following table: µg of mRNA (from step 1)

≤1 2 3 4 5 SuperScript® II RT (µL) 1 2 3 4 5

Mix gently and incubate at 37°C for 1 hour. Regardless of the amount of starting mRNA, the total volume should now be 20 µL.

Final composition of the reaction: 50 mM Tris-HCl (pH 8.3) 75 mM KCl 3 mM MgCl2 10 mM DTT 500 µM each dATP, dCTP, dGTP, and dTTP 50 µg/mL oligo(dT) 12-18 primer and/or 2.5–7.5 µg/mL random

hexamers ≤5 µg (≤250 µg/mL) mRNA 10,000–50,000 units/mL SuperScript® II RT

5. Place the tube on ice to terminate the reaction.6. Remove 2 µL from the reaction and add it to a microcentrifuge tube containing

43 µL of 20 mM EDTA (pH 7.5) and 5 µL of Yeast tRNA. This mixture will be used in calculating first-strand yield.

7. Take the remaining 18 µL of the first-strand reaction and continue immediately with the first two steps of the second-strand reaction as described in Section 3.4.

8. While the second-strand reaction is incubating, spot duplicate 10-µL aliquots from the diluted sample from step 6 of this section onto glass fiber filters. Dry one of the filters under a heat lamp or at room temperature. This filter will be used to determine the specific activity of the dCTP reaction.

9. Wash the other filter three times in sequence, for 5 minutes each time, in a beaker containing 50 mL of fresh, ice-cold 10% (w/v) TCA containing 1% (w/v) sodium pyrophosphate. Wash the filter once with 50 mL of 95% ethanol at room temperature for 2 minutes. Dry the filter under a heat lamp or at room temperature. This filter will be used to determine the yield of first-strand cDNA.

10. Count both filters in standard scintillant to determine the amount of 32P in the reaction, as well as the amount of 32P that was incorporated. See Section 3.11, Analysis of cDNA Products, for information needed to convert the data into yield of first-strand cDNA.

16

11. Precipitate the remaining 30 µL of the sample from step 6 of this section by adding 15 µL of 7.5 M NH4OAc, followed by 90 µL of absolute ethanol (–20°C). Vortex the mixture thoroughly and immediately centrifuge at room temperature for 20 minutes at 14,000 × g.

12. Remove the supernatant carefully, and overlay the pellet with 0.5 mL of 70% ethanol (–20°C). Centrifuge for 2 minutes at 14,000 × g and remove the supernatant.

13. Dry the cDNA at 37°C for 10 minutes to evaporate residual ethanol and proceed to Section 3.11, Analysis of cDNA Products.

3.4 Second-Strand SynthesisThis protocol is suitable for synthesizing second-strand cDNA from ≤5 µg mRNA originally in the 20-µL first-strand reaction.1. On ice, add the following reagents, in the order shown, to the first-strand

reaction tube:Component Volume (µL)DEPC-Treated Water ......................................................................................935X Second-Strand Buffer ................................................................................3010 mM dNTP Mix ..............................................................................................3E. coli DNA Ligase (10 units/µL) .......................................................................1E. coli DNA Polymerase I (10 units/µL) ............................................................4E. coli RNase H (2 units/µL) .............................................................................1Final volume .................................................................................................150

Final composition of the reaction: 25 mM Tris-HCl (pH 7.5) 100 mM KCl 5 mM MgCl2 10 mM (NH4)2SO4

0.15 mM ß-NAD+

250 µM each dATP, dCTP, dGTP, dTTP 1.2 mM DTT 65 units/mL DNA ligase 250 units/mL DNA polymerase I 13 units/mL RNase H

2. Vortex the tube gently to mix and incubate the completed reaction for 2 hours at 16°C. Do not let the temperature rise above 16°C.

3. Add 2 µL (10 units) of T4 DNA Polymerase and continue incubating at 16°C for 5 minutes.

4. Place the reaction on ice and add 10 µL of 0.5 M EDTA. Note: If [α-32P]dCTP was added to the second-strand reaction, remove 10 µL from the reaction, and add it to a microcentrifuge tube containing 35 µL of 20 mM EDTA (pH 7.5) and 5 µL of Yeast tRNA. This mixture will be used in calculating second-strand yield. Then proceed as described in steps 8 to 10 in Section 3.3 for processing and counting the filters.

5. Add 150 µL of phenol:chloroform:isoamyl alcohol (25:24:1), vortex thoroughly, and centrifuge at room temperature for 5 minutes at 14,000 × g to separate the phases. Carefully remove 140 µL of the upper, aqueous layer, and transfer it to a fresh 1.5-mL microcentrifuge tube.

6. Add 70 µL of 7.5 M NH4OAc, followed by 0.5 mL of absolute ethanol (–20°C). Vortex the mixture thoroughly and immediately centrifuge at room temperature for 20 minutes at 14,000 × g.

7. Remove the supernatant carefully and overlay the pellet with 0.5 mL of 70% ethanol (–20°C). Centrifuge for 2 minutes at 14,000 × g and remove the supernatant.

8. Dry the cDNA at 37°C for 10 minutes to evaporate residual ethanol and proceed to Section 3.5.

Caution: If the first-strand cDNA was labeled, the supernatant(s) wil l be radioactive. Dispose of this material properly.

Caution: If the first or second-strand cDNA was labeled, the supernatant(s) will be radioactive. Dispose of this material properly.

3

17

3.5 EcoRI (Not I) Adapter Addition1. Add the following reagents on ice, in the order shown, to the cDNA from step 8

of Section 3.4.Component Volume (µL)DEPC-treated Water .......................................................................................185X Adapter Buffer ...........................................................................................10EcoR I (Not I) Adapters...................................................................................100.1 M DTT .........................................................................................................7T4 DNA Ligase .................................................................................................5Final volume ...................................................................................................50

Final composition of the reaction: 66 mM Tris-HCl (pH 7.6)

10 mM MgCl2 1 mM ATP 14 mM DTT 200 µg/mL EcoR I (Not I) adapters 100 units/mL T4 DNA ligase

2. Mix gently and incubate the reaction at 16°C for a minimum of 16 hours. For greatest convenience, simply let the reaction proceed overnight.

3. Heat the reaction at 70°C for 10 minutes to inactivate the ligase.4. Place the reaction on ice and proceed to Section 3.6.

3.6 Phosphorylation of EcoR I-Adapted cDNA1. Add 3 µL of T4 Polynucleotide Kinase to the reaction from step 4 of Section

3.5.2. Mix gently and incubate the reaction for 30 minutes at 37°C. Note: While the

reaction is incubating, you may begin performing steps 1 through 3 of Section 3.7.

3. Heat the reaction at 70°C for 10 minutes to inactivate the kinase.4. Place the reaction on ice and proceed to Section 3.7.

3.7 Column ChromatographyThis procedure optimizes size fractionation of the cDNA and makes the cloning of larger inserts more probable. This procedure also ensures that residual adapters do not enter into the library. Failure to adhere to these instructions can compromise the quality of your cDNA library.1. Place one of the cDNA size fractionation columns in a support. Remove the

top cap first, and then the bottom cap. Allow the excess liquid (20% ethanol) to drain.

2. Pipet 0.8 mL of TEN buffer [10 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 25 mM NaCl; autoclaved] onto the upper frit and let it drain completely. Repeat this step three more times for a total of 3.2 mL. Each 0.8-mL wash will take approximately 15 minutes, but it is important to do all four washes to remove the 20% ethanol from the column.

3. Label 20 sterile microcentrifuge tubes from 1 to 20, and place them in a rack with tube 1 under the outlet of the column.

4. Add 97 µL of TEN buffer to the cDNA reaction from step 4 of Section 3.6 and mix gently.

5. Add the entire sample to the center of the top frit and let it drain into the bed. Collect the effluent into tube 1.

6. Add 100 µL of TEN buffer to the column and collect the effluent into tube 2. Note: Let the column drain completely—in other words, until it stops dripping—before the addition of each new 100-µL aliquot.

7. Beginning with the next 100-µL aliquot of TEN buffer, collect single-drop (~35 µL) fractions into individual tubes. Continue adding 100-µL aliquots of TEN buffer until you have collected a total of 18 drops into tubes 3 through 20, one drop per tube.

Note: Do not add more than one reaction per column. Addition of multiple reactions will result in poor cloning efficiency.

Note: If the flow rate is noticeably slower than 20 min/mL, do not use the column. Additionally, if the drop size from the column is not ~25 to 35 µL, do not use the column. The integrity and resolution of the cDNA might be compromised.

Tip: When collecting fractions, wear gloves that have been rinsed with ethanol to reduce static. Also, position the tube 1 to 2 cm from the bottom of the column to avoid the effects of static on drop size.

8. Using an automatic pipet, measure the volume in each tube; use a fresh tip for each fraction to avoid cross-contamination. Record each value in column A of one of the following work tables, using the sample table as a guide. Cap each tube after the volume has been measured and recorded. Calculate the cumulative elution volume with the addition of each fraction and record this value in column B. Note: The sample table contains data typical for the size fractionation protocol when oligo(dT) has been used to prime first-strand cDNA synthesis; actual data will vary. The sample is provided merely to illustrate the decision-making process for selecting cDNA for the vector ligation reaction.

9. Identify the fraction for which the value in column B is closest to, but not exceeding, 600 µL (corresponding to fraction 12 in the sample table). Draw a horizontal line across the table immediately below this fraction. Do not use any of the subsequent fractions for your cDNA library; remove them to a separate tube rack to avoid accidentally using them in the remainder of the protocol. Important: Fractions collected after the 600-µL cutoff point (corresponding to tubes 13 through 20 in the sample table) will contain smaller cDNAs and unligated adapters. Use of these fractions significantly increases the risk of cloning the EcoR I (Not I) adapters. In some cases with random hexamer-primed reactions, the target cDNA may elute in a later fraction, in which case taking fractions beyond the passage of 600 µL may be a necessary risk.

10. Place the remaining tubes in a scintillation counter and obtain Cerenkov counts for each fraction. Count the entire sample in the tritium channel; do not add scintillation fluid to the tubes. In column C, record the counts corresponding to each fraction. Note: Cerenkov counts above background should appear after passage of 450–500 µL of buffer.

11. For each fraction in which the Cerenkov counts exceed background (corresponding to fractions 8 to 12 in the sample table), calculate the amount of cDNA, using equation 5 in Section 3.12, Analysis of cDNA from the cDNA Size Fractionation Column. Record each cDNA amount in column D.

12. Divide each cDNA amount in column D by the fraction volume given in column A to determine the cDNA concentration per fraction. Record this value in column E.

13. The plasmid vector ligation reaction in Section 3.8 requires 10 ng of cDNA at ≥1 ng/µL. The λ vector ligation in Section 3.9 requires 50 ng of cDNA as a dried pellet. Examine the data entered in columns D and E of your work table and decide which fractions to pool and precipitate early fractions, or (if applicable), to use the appropriate amount of cDNA from a suitable fraction directly in the selected vector ligation reaction. Guidelines for making this decision are provided in Section 3.12, Analysis of cDNA from the cDNA Size Fractionation Column.

14. If cDNA from two or more fractions must be pooled to obtain the cDNA needed for the vector ligation reaction, uncap the first selected tube (corresponding to fraction 8 in the sample table), and add cDNA from each subsequent fraction until there is the correct amount of cDNA (as determined in step 13) in the tube. Measure the volume and add 5 µL of Yeast tRNA to the tube.

15. Add 0.5 volumes of NH4OAc, followed by 2 volumes of absolute ethanol (–20°C). Vortex the mixture thoroughly and immediately centrifuge at room temperature for 20 minutes at 14,000 × g. Note: If you are stopping here (see “Time Planning” under Section 3.2, General Comments), store the tubes at –20°C overnight before centrifugation to minimize the effects of 32P decay.

16. Remove the supernatant carefully and overlay the pellet with 0.5 mL of 70% ethanol (–20°C). Centrifuge for 2 minutes at 14,000 × g and remove the supernatant.

17. Dry the cDNA at 37°C for 10 minutes to evaporate residual ethanol. To ligate to a plasmid vector, dissolve the cDNA in 10 µL of TEN buffer and proceed to Section 3.8. For ligation to a λ vector, proceed directly to Section 3.9.

Methods

18

19

1 150 150 25

2 97 247 30

3 34 281 32

4 30 311 20

5 35 346 29

6 33 379 32

7 34 413 43

8 34 447 125 3.3 0.1

9 36 483 625 16 0.44

10 34 517 1,196 32 0.94

11 34 551 1,740 46 1.4

12 34 585 1,523 40 1.2

13 34 619

14 30 649

15 33 682

16 35 717

17 32 749

18 36 785

19 34 819

20 35 854

No.

A

FractionVolume

(µL)

B

TotalVolume

(µL)

C

CerenkovCounts(CPM)

D

Amount of cDNA

(ng)

EConcen-tration of

cDNA(ng/µL)

Sample Experiment

No.

A

FractionVolume

(µL)

B

TotalVolume

(µL)

C

CerenkovCounts(CPM)

D

Amount of cDNA

(ng)

EConcen-tration of

cDNA(ng/µL)

Experiment 1

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

3

20

Methods

No.

A

FractionVolume

(µL)

B

TotalVolume

(µL)

C

CerenkovCounts(CPM)

D

Amount of cDNA

(ng)

EConcen-tration of

cDNA(ng/µL)

Experiment 2

No.

A

FractionVolume

(µL)

B

TotalVolume

(µL)

C

CerenkovCounts(CPM)

D

Amount of cDNA

(ng)

EConcen-tration of

cDNA(ng/µL)

Experiment 3

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

3

21

3.8 Ligation of cDNA to a Plasmid VectorIf you intend to ligate the cDNA to a λ vector, use Section 3.9 or 3.10. This protocol is intended for use with 10 ng of cDNA. Note: Do not proceed with this protocol until you have made the appropriate decisions regarding the choice of fractions for use in the ligation reaction. For more information, refer to Section 3.12, Analysis of cDNA from the cDNA Size Fractionation Column.

1. Add the following, at room temperature, to a sterile 1.5-mL microcentrifuge tube:Component Amount5X T4 DNA Ligase Buffer ............................................................................4 µLplasmid vector, EcoR I-cut, dephosphorylated (50 ng/µL) .........................50 ngcDNA (≥1 ng/µL) ........................................................................................10 ngDEPC-Treated Water ..............................sufficient to bring the volume to 19 µL

2. Add 1 µL of T4 DNA Ligase and mix by pipetting. Final composition of the reaction:

50 mM Tris-HCl (pH 7.6) 10 mM MgCl2 1 mM ATP 5% (w/v) PEG 8000 1 mM DTT 2.5 µg/mL plasmid vector, EcoR I-Cut 0.5 µg/mL cDNA 50 units/mL T4 DNA ligase

3. Let the reaction incubate for 3 hours at room temperature or overnight at 4°C. Note: Following incubation, the cDNA will be ligated into the cloning vector and ready for transformation into E.coli cells such as MAX Efficiency® DH5α™ or DH10B™ Competent Cells, or for precipitation procedures prior to electroporation into cells such as ElectroMAX™ DH10B Cells. See Section 6 for information on these products.

3.9 LigationofcDNAtoλgt11andλgt10VectorsIf you intend to ligate the cDNA to a plasmid vector, use Section 3.8. This protocol is intended for use with 50 ng of cDNA as a dried pellet. Note: Do not proceed with this protocol until you have made the appropriate decisions regarding the choice of fractions for use in the ligation reaction. For more information, refer to Section 3.12, Analysis of cDNA from the cDNA Size Fractionation Column.

1. Add the following, at room temperature, to a sterile 1.5-mL microcentrifuge tube containing the dried cDNA pellet:Component Amount5X T4 DNA Ligase Buffer .............................................................................1 µLλ vector, EcoR I Arms (250 ng/µL) ............................................................500 ngcDNA (as a dried pellet)..............................................................................50 ngDEPC-treated water .................................. sufficient to bring the volume to 4 µL

Mix by pipetting to ensure that the cDNA is completely dissolved.2. Add 1 µL of T4 DNA Ligase and mix by pipetting. Final composition of the reaction:

50 mM Tris-HCl (pH 7.6) 10 mM MgCl2 1 mM ATP 5% (w/v) PEG 8000 1 mM DTT 100 µg/mL λ vector, EcoR I Arms 10 µg/mL cDNA 200 units/mL T4 DNA ligase

Note: Although 10 to 20 ng of cDNA generally saturates the vector, the amount of cDNA that yields the maximal number of clones may be higher (e.g., 40 ng). As much as 14 µL of cDNA in TEN buffer may be added to the ligation reaction.

Note: Although 20 to 50 ng of cDNA generally saturates the vector, the amount of cDNA can be varied to determine what quantity yields the maximal number of clones.

3. Let the reaction incubate for 3 hours at room temperature or overnight at 4°C. Note: Following incubation, the cDNA will be ligated into the cloning vector and ready for in vitro packaging.

3.10LigationofcDNAtoaλZipLoxVector1. Prepare a 5X DNA ligase buffer separately. Do not use the 5X T4 DNA Ligase

Buffer supplied with this system; it contains PEG which inhibits the packaging of λZipLox. Mix 10 µL of the 10X DNA Ligase Buffer, supplied with the λZipLox [400 mM Tris-HCl (pH 7.5), 100 mM MgCl2, 15 mM ATP] with 10 µL of 100 mM DTT in a microcentrifuge tube on ice.

2. Add the following, at room temperature, to a sterile 1.5-mL microcentrifuge tube containing the dried cDNA pellet:Component Amount5X DNA Ligase buffer (from step 1) ..............................................................1 µLλZipLox Arms, Not I - Sal I (250 ng/µL) ....................................................... 2 µLcDNA (as a dried pellet).....................................................................20 to 50 ngdistilled water ................................................................................................1 µL

Mix by pipetting to ensure that the cDNA is completely dissolved.3. Add 1 µL (one unit) of T4 DNA ligase and mix gently by pipetting.4. Let the reaction incubate for 3 hours at room temperature or overnight at 4°C.5. Following incubation, the cDNA will be ligated into the cloning vector and ready

for in vitro packaging.

3.11 Analysis of cDNA Products3.11.1 First-Strand YieldThe overall yield of the first-strand reaction is calculated from the amount of acid-precipitable radioactivity determined as described in Section 3.3. In order to perform the calculation, you must first determine the specific activity (SA) of the radioisotope in the reaction. The specific activity is defined as the counts per minute (cpm) of an aliquot of the reaction divided by the quantity (in pmol) of the same nucleotide in the aliquot. For [α-32P]dCTP, the specific activity is given by the relationship:

cpm/10 µL SA (cpm/pmol dCTP) = [1] 200 pmol dCTP/10 µL

The amount of dCTP contributed by the radiolabeled material is insignificant relative to the unlabeled nucleotide and is ignored in equation 1.

Once the specific activity is known, the amount of cDNA in the first-strand reaction can be calculated from the amount of acid-precipitable radioactivity determined from the washed filter:

Amount of (cpm) × (50 µL/10 µL) × (20 µL/2 µL) × (4 pmol dNTP/pmol dCTP) = [2] ds cDNA (µg) (cpm/pmol dCTP) × (3,030 pmol dNTP/µg cDNA)

The correction in the numerator takes into account that, on the average, four nucleotides will be incorporated into the cDNA for every dCTP scored by this assay. The factor in the denominator is the amount of nucleotide that corresponds to 1 µg of single-stranded DNA.

Example: The unwashed filter gave 50,000 cpm when it was counted. The specific activity of the dCTP is given by equation 1:

50,000 cpm/10 µL SA (cpm/pmol dCTP) = 200 pmol dCTP/10 µL

= 250 cpm/pmol dCTP

22

Note: Although 20 to 50 ng of cDNA generally saturates the vector, the amount of cDNA can be varied to determine what quantity yields the maximal number of clones.

Methods

23

If 2 µg of starting mRNA was used and the washed filter gave 1,800 cpm, then the amount of cDNA is calculated using equation 2:

Amount of (1,800 cpm) × (50 µL/10 µL) × (20 µL/2 µL) × (4 pmol dNTP/pmol dCTP) = ds cDNA (µg) (250 cpm/pmol dCTP) × (3,030 pmol dNTP/µg cDNA)

= 0.5 µg first-strand cDNA

This amount of first-strand cDNA would represent a 25% yield relative to the 2 µg of mRNA starting material.

3.11.2 Second-Strand YieldThe overall yield of the second-strand reaction is calculated from the amount of acid-precipitable radioactivity determined as described in Section 3.4. In order to perform the calculation, you must first determine the specific activity of the radioisotope in the reaction. The specific activity is defined as the counts per minute of an aliquot of the reaction divided by the quantity of the same nucleotide in the aliquot. For [α-32P]dCTP, the specific activity is given by the relationship:

cpm/10 µL SA (cpm/pmol dCTP)= [3] 500 pmol dCTP/10 µL

The amount of dCTP contributed by the radiolabeled material is insignificant relative to the unlabeled nucleotide and is ignored in equation 3.

Once the specific activity is known, the amount of cDNA in the second-strand reaction can be calculated from the amount of acid-precipitable radioactivity determined from the washed filter:

Amount of (cpm) × (50 µL/10 µL) × (150 µL/10 µL) × (4 pmol dNTP/pmol dCTP) = [4] cDNA (µg) (cpm/pmol dCTP) × (3,030 pmol dNTP/µg cDNA)

The correction in the numerator takes into account that, on the average, four nucleotides will be incorporated into the cDNA for every dCTP scored by this assay. The factor in the denominator is the amount of nucleotide that corresponds to 1 µg of single-stranded DNA.

Example: The unwashed filter gave 300,000 cpm when it was counted. The specific activity of the dCTP is given by equation 3:

300,000 cpm/10 µL SA (cpm/pmol dCTP)= 500 pmol dCTP/10 µL

= 600 cpm/pmol dCTP

If 2 µg of starting mRNA was used and the washed filter gave 2,500 cpm, then the amount of cDNA is calculated using equation 4:

Amount of (2,500 cpm) × (50 µL/10 µL) × (150 µL/10 µL) × (4 pmol dNTP/pmol dCTP) = cDNA (µg) (600 cpm/pmol dCTP) × (3,030 pmol dNTP/µg cDNA)

= 0.4 µg second-strand cDNA

3.11.3 Gel AnalysisThe first or second-strand cDNA, if labeled with 32P, can be analyzed by alkaline agarose gel electrophoresis to estimate the size range of products synthesized (15,28).

The ethanol-precipitated first-strand sample is dissolved in 10 µL 1X alkaline agarose gel sample buffer [30 mM NaOH, 1 mM EDTA, 10% (v/v) glycerol, 0.01% bromophenol blue]. Other samples (such as the 1 Kb DNA Ladder, labeled with 32P) can be electrophoresed after addition of a suitable volume of a more concentrated sample buffer; the only precaution is to chelate any Mg2+ by addition of EDTA prior to adding the alkaline sample buffer.

3

The gel [1.4% (w/v)] should be cast in the appropriate volume of 30 mM NaCl, 2 mMEDTA (alkaline buffer cannot be used because it will degrade the agarose when the solution is microwaved to melt the agarose) and should be equilibrated for 2 to 3 hours in alkaline electrophoresis buffer (30 mM NaOH, 2 mM EDTA) before loading the samples. Electrophoresis should be for 5 to 6 hours at 50 V or for 16 to 18 hours at 15 V. The gel should be dehydrated under vacuum until the buffer is removed, then under heat and vacuum for several hours to complete the drying. The dried gel should then be exposed to x-ray film overnight at room temperature.

When a heterogeneous mRNA population is fractionated by alkaline gel electrophoresis, a continuum of fragments ranging in size from 500 to 5,000 nucleotides makes up the bulk of the first and second-strand cDNA. Figure 5 shows an alkaline electrophoretic analysis of 32P-labeled first and second-strand cDNA synthesized from oligo(dT)-primed HeLa mRNA with the SuperScript® Choice System.

3.12 Analysis of cDNA from the cDNA Size Fractionation ColumnCalculation of the amount of size-fractionated cDNA in each column fraction is necessary to ensure that the proper decisions are made concerning fraction selection and that the cDNA is used economically in the ligation reaction. The Cerenkov counts are approximately 50% of the radioactivity that would be measured in scintillant. The counts are converted into nanograms of cDNA using the specific activity determined in Section 3.3 or 3.4. The amount of cDNA (as double strand) in each fraction is given by the following relationship:Amount of (Cerenkov cpm) × 2 × (4 pmol dNTP/pmol dCTP) × (1,000 ng/µg ds cDNA) = [5]ds cDNA (ng) SA (cpm/pmol dCTP) × (1,515 pmol dNTP/µg ds cDNA)

Example: If one of the fractions from the column gave 1,500 cpm when counted by Cerenkov radiation (first-strand-labeled), then the amount of cDNA in that fraction is calculated using equation 5:Amount of (1,500 cpm) × 2 × (4 pmol dNTP/pmol dCTP) × (1,000 ng/µg ds cDNA) = ds cDNA (ng) SA (cpm/pmol dCTP) × (1,515 pmol dNTP/µg ds cDNA)

= 32 ng

After calculating the amount and the concentration of cDNA in each fraction (columns D and E of the work table), you are ready to select and recover cDNA for use in the vector ligation reaction in Section 3.8, 3.9, or 3.10. Depending on which type of vector you intend to use, certain considerations should be noted:

Recovering cDNA for ligation to plasmid vectors. If you wish to maximize the average insert size in a plasmid-based cDNA library and your earliest selected fraction (corresponding to fraction 8 in the sample table) contains less than the 10 ng of cDNA required for Section 3.8, you will need to pool cDNA from this fraction with at least a portion of subsequent fractions. Because the resulting cDNA solution will be too dilute for use in the ligation reaction, you will also need to ethanol-precipitate the pooled cDNA. For example, fraction 8 from the sample table in Section 3.7 contains 3.3 ng of cDNA in 34 µL, and fraction 9 contains 16 ng of cDNA in 36 µL. If all of fraction 8 is combined with 15 µL from fraction 9, the pool will contain 10 ng of cDNA in 49 µL. The ethanol precipitation steps in Section 3.7 will then concentrate the 10 ng cDNA in preparation for the plasmid vector ligation reaction in Section 3.8.

If any of the selected fractions contain ≥10 ng of cDNA at ≥1 ng/µL, you can use 10 ng from the fraction directly in the plasmid vector ligation reaction in Section 3.8. Note: If this fraction is not the earliest selected, based on Cerenkov counts (corresponding to fraction 8 in the sample table), the average insert size in your cDNA library will be smaller than could be obtained through additional pooling and

24

Methods

Figure 5. Alkaline agarose gel analy-sis of first and second-strand cDNA synthesized with the SuperScript® Choice System. Samples of 32P-labeled first or second-strand cDNA made from HeLa mRNA were ethanol-precipitated, dissolved in alkaline agarose sample buffer, and electrophoresed on a 1.4% agarose gel at 15 V for 16 h.

Mar

ker

Firs

t-Str

and

Seco

nd-S

tran

d

kb

7.1 6.1 5.1

4.0

3.0

2.0

1.6

1.0

0.5

25

ethanol precipitation steps. Figure 6 provides an electrophoretic display of the size ranges of cDNA obtained in fractions 8 through 19 in the experiment that generated the sample table data.

Recovering cDNA for ligation to λ vectors. If you wish to maximize the average insert size in a λ-based cDNA library and your earliest selected fraction (corresponding to fraction 8 in the sample table) contains less than the 50 ng of cDNA required for Section 3.9 or 3.10, you will need to pool cDNA from this fraction with at least a portion of subsequent fractions. Because the cDNA for use in Section 3.9 or 3.10 must be in the form of a dry pellet, you will need to ethanol-precipitate the cDNA, pooled or not. For example, pooling all of fractions 8, 9, and 10 from the sample table yields ~50 ng of cDNA in solution; the ethanol precipitation steps in Section 3.7 will then concentrate the cDNA, and 50 ng from the sample can be used in the λ vector ligation reaction in Section 3.9 or 3.10.

The preceding discussion assumes that you wish to achieve maximum transformation efficiencies in keeping with the design of Section 3.8, 3.9, or 3.10. One other procedural option is available: if you wish merely to maximize insert size and are willing to accept lower transformation efficiencies to attain this goal, you may use your earliest selected fraction in Section 3.8, 3.9, or 3.10 even if it contains less than the required amount of cDNA.

Please note that the above option should preferably be attempted in plasmid vectors using electroporation methods, or in λ vectors using in vitro packaging methods, since these methods offer higher cloning efficiencies (and thus a greater chance of generating a sufficiently large cDNA library).

3

Figure 6. Electrophoretic analysis of size-fractionated cDNA. [32P]cDNA was fractionated on a 1-mL prepacked column equilibrated in TEN buffer. Single-drop fractions (~35 µL each) were collected, and aliquots were analyzed by electrophoresis on a 1% agarose gel in 40 mM Tris-acetate (pH 8.3), 5 mM sodium acetate, 1 mM EDTA. The gel was electrophoresed at 200 V for 2 hours.

kb

12.2

7.1

5.1

3.0

2.0

1.6

1.0

Column Fraction 8 9 10 11 12 13 14 15 16 17 18

4

26

4.1 Isolation of mRNAIn the first step of cDNA library construction, RT converts the sequence information of the mRNA to first-strand cDNA. The quality of the mRNA used as the template will influence profoundly the yield and size distribution of the first-strand product. We recommend a guanidine isothiocyanate-based homogenization procedure to ensure rapid inactivation of RNases and general deproteinization, followed by two selections of mRNA by oligo(dT) cellulose chromatography to enrich maximally for poly(A)+ RNA.

The mRNA preparation can be analyzed by formaldehyde agarose gel electrophoresis (16,29) and ethidium bromide staining (17,30). The gel should reveal a smear of fluorescent material, and perhaps some discrete fragments corresponding to abundant mRNAs. Residual 18S or 28S rRNAs (approximately 2,000 or 5,000 bases in length, respectively, in mammalian cells) will be visible even in mRNA preparations highly enriched for poly(A)+ RNA, and will be indicative of an intact mRNA population. If neither rRNA is visible and the distribution of the mRNA is not centered in the 1- to 3-kb range, then you will need to consider troubleshooting your RNA isolation procedure. Examples of representative mRNA preparations have been published (31).

4.2 First-Strand Reaction The conditions described for the first-strand reaction have been optimized; thus it is imperative to follow Section 3.3 explicitly. Do not increase the size of the first-strand reaction from 20 µL.

First-strand yields will vary widely; using 2 µg of mRNA in the first-strand reaction, we routinely obtain 25% to 35% yields from HeLa mRNA preparations primed with oligo(dT)12-18 and 25% to 60% yields from the HeLa mRNA preparations primed with 50 to 150 ng random hexamers, either by themselves or in combination with oligo(dT). Analysis of the products by alkaline gel electrophoresis reveals a distribution from 0.5 to >7 kb (see figure 5). A lower yield does not necessarily indicate that a library cannot be made, so long as the size distribution of the products is consistent with the size distribution of the mRNA. A lower size distribution, if the reaction is primed with oligo(dT), suggests RNase contamination; however, some random hexamer-primed reactions may inherently result in a lower size distribution due to multiple priming starts throughout the RNA template. If RNase contamination is suspected, use the control RNA to synthesize first-strand cDNA and examine the products by alkaline gel electrophoresis.

If you obtain a poor yield in the first-strand reaction, the control RNA can be used to verify that the system components are working properly. The control RNA is 2.0-kb single transcript that can be substituted directly into the first-strand reaction as described in Section 3.3 [use of the extra components provided (see Section 3.2.4, Utilization of Reagents) allows you to reverse transcribe the control RNA without losing the capability to construct three cDNA libraries as described in Sections 3.3 through 3.9]. The first-strand yield using the control RNA is generally 30% to 50%. If desired, the control RNA can be taken through the entire procedure and cloned into the vector of choice.

Troubleshooting

Note: TRIzol® Reagent can be used to isolate high quality total RNA from cells and tissue.

4

27

4.3 Second-Strand ReactionThe second-strand reaction in Section 3.4 is not sensitive to RNase contamina tion, but strict adherence to good laboratory practice is still required. The second-strand reaction is generally efficient, and yields of 80% to 100% (relative to the amount of first-strand cDNA synthesized) are common. The distribution of second-strand cDNA products should look like the distribution of the first-strand cDNA products when analyzed by alkaline agarose gel electrophoresis (see Figure 5).Unlike some second-strand procedures that do not require E. coli RNase H (13), this enzyme must be included to provide initiation points for nick translation by DNA polymerase I when first-strand cDNA is synthesized by SuperScript® II RT. Furthermore, the reaction must be incubated at ≤16°C to prevent spurious synthesis by DNA polymerase I, although this is contrary to the original descriptions in the literature (13,14).Dilution of the first-strand reaction precisely as specified in Section 3.3 is extremely important because the pH of the second-strand reaction differs from that of the first-strand reaction. This pH change influences the activity of the 3´→5´ and 5´→3´ exonucleases of DNA polymerase I, thereby limiting the number of nucleotides that are removed from the termini of the cDNA as the second strands are completed.

4.4 EcoR I (Not I) Adapter AdditionThis protocol is the most difficult to troubleshoot because it will not be suspect until the cloning efficiency is determined. The adapter addition step is assayed most readily by nondenaturing polyacrylamide gel electrophoresis. If a 10-µL aliquot of the adapter ligation reaction is electrophoresed on a 12% acrylamide gel and the DNA is visualized by ethidium bromide staining, the ligation of two adapters to each other at their blunt ends will be evidenced by a conspicuous 38-bp fragment. Although this does not verify that the adapters have ligated to the cDNA, it does show that the ligation reaction in Section 3.5 is functionally sound.

4.5 Phosphorylation of EcoR I-Adapted cDNAProblems with phosphorylation will not be apparent until the cloning efficiency is determined. To test the phosphorylation protocol, perform a control reaction as follows: 1. Add the following to a sterile 1.5-mL microcentrifuge tube:

Component Volume (µL)5X Adapter Buffer .............................................................................................3EcoR I (Not I) Adapters.....................................................................................1DEPC-Treated Water ........................................................................................70.1 M DTT .........................................................................................................2[γ-32P]ATP (5 µCi/µL) ........................................................................................1T4 Polynucleotide Kinase .................................................................................1Final volume ...................................................................................................15

2. Mix gently and incubate the reaction at 37°C for 30 minutes.3. Heat the reaction at 70°C for 10 minutes to inactivate the kinase.4. Spot duplicate 1-µL aliquots on DE-81 ion-exchange paper. Dry one filter to

determine the amount of isotope added. Wash the other filter twice in 0.3 M ammonium formate at room temperature to remove unincorporated isotope, rinse with water, and allow to dry.

28

5. Count both samples in standard fluor and determine the percentage of adapters phosphorylated as follows:

% adapters phosphorylated = cpm of washed filter × 100 0.006 × cpm of unwashed filter

You should obtain 70% to 100% phosphorylation of the EcoR I (Not I) adapters by this assay. Caution: If 32P-labeled adapters are substituted for unlabeled adapters in Section 3.5, you will be unable to quantitate the amount of DNA recovered after column chromatography, as described in Section 3.12, Analysis of cDNA from the cDNA Size Fractionation Column. Use 32P-labeled adapters only to troubleshoot Section 3.6.

4.6 Column ChromatographyThe most likely problems with Section 3.7 will arise either from running the column too quickly—it is imperative to let it go dry between 100-µL aliquots—or taking fractions beyond 600 µL. If fractions beyond 600 µL are used, the library will contain more small cDNAs (100–500 bp) or apparently “empty” clones. The empty clones arise from ligation of the EcoR I (Not I) adapters. Please refer to Section 3.12, Analysis of cDNA from the cDNA Size Fractionation Column and the sample table in Section 3.7 for representative size fractionation results and for guidelines on selecting the proper fractions.

4.7 Ligation of cDNA to a Plasmid VectorIf the transformation efficiency is low, the plasmid ligation reaction in Section 3.8 may not be proceeding properly. If the remainder of the ligation reaction is electrophoresed on an agarose gel (along with 1 µL of the vector as a control, run in an adjacent lane) and visualized by ethidium bromide staining (using a strong 254-nm UV transluminator), the DNA should smear upward from the position of the vector. If the DNAs did not ligate, repeat Section 3.8 using a smaller volume (for example, 2 µL) of the column fraction containing the highest concentration of cDNA; however, do not go beyond the first five fractions that contain cDNA. This procedure will minimize inhibition if the column buffer is a problem, although up to 14 µL of the column buffer generally can be used in the ligation.

Another cause of low transformation efficiencies is using an insufficient amount of cDNA in the ligation reaction. Recheck all calculations used to generate the data for the table to rule out the possibility of a simple mathematical error. If the described ligation conditions do not yield enough transformants to make your library complete, the vector-to-cDNA ratio yielding the maximum number of clones should be determined empirically. Also, if you chose not to ethanol-precipitate the cDNA, when it would have been the recommended procedure following size fractionation, return to step 13 of Section 3.7 to ethanol-precipitate the cDNA and proceed to Section 3.8.

4.8 LigationofcDNAtoaλVectorIf the in vitro packaging efficiency is low, the λ ligation reaction in Section 3.9 or 3.10 may not be proceeding properly. If 2 µL of the ligation reaction is electrophoresed on an agarose gel (along with 1 µL of the vector as a control, run in an adjacent lane) and visualized by ethidium bromide staining (using a strong 254-nm UV transluminator), the DNA should smear upward from the position of the λ vector arms. If the DNA did not ligate, repeat Section 3.9 or 3.10.

Again, recheck all calculations to rule out the possibility of an insufficient amount of cDNA in the ligation reaction.

Troubleshooting

5

29

References

1. Aviv, H. and Leder, P. (1972) Proc. Natl. Acad. Sci. USA 69, 1408.2. Binns, M.M., Boursnell, M.E.G., Foulds, I.J., and Brown, T.D.K. (1985) J. Virol.

Methods 11, 265.3. Ozkaynak, E., Rueger, D.C., Drier, E.A., Corbett, C., Ridge, R.J., Sampath, T.K.,

and Oppermann, H. (1990) EMBO J. 9, 2085.4. Birnstiel, M.L., Busslinger, M., and Strub, K. (1985) Cell 41, 349.5. Dowling, P.C., Giorgi, C., Roux, L., Dethlefsen, L.A., Galantowicz, M.E., Blumberg,

B.M., and Kalakofsky, D. (1983) Proc. Natl. Acad. Sci. USA 80, 5213.6. Gruber, C.E., Cain, C., and D'Alessio, J.M. (1991) Focus® 13, 88.7. Kotewicz, M.L., D’Alessio, J.M., Driftmeir, K.M., Blodgett, K.P., and Gerard, G.F.

(1985) Gene 35, 249.8. Kotewicz, M.L., Sampson, C.M., D’Alessio, J.M., and Gerard, G.F. (1988) Nucl.

Acids Res. 16, 265.9. Berger, S.L., Wallace, D.M., Puskas, R.S., and Eschenfeldt, W.H. (1983)

Biochemistry 22, 2365.10. Gerard, G.F., D’Alessio, J.M., and Kotewicz, M.L. (1989) Focus 11, 66.11. D’Alessio, J.M., Gruber, C.E., Cain, C., and Noon, M.C. (1990) Focus 12, 47.12. Gerard, G.F., Schmit, B.J., Kotewicz, M.L., and Campbell, J.H. (1992) Focus 14,

91.13. Okayama, H. and Berg, P. (1982) Mol. Cell. Biol. 2, 161.14. Gubler, U. and Hoffman, B.J. (1983) Gene 25, 263.15. D’Alessio, J.M. and Gerard, G.F. (1988) Nucl. Acids Res. 16, 1999.16. Huynh, T.V., Young, R.A., and Davis, R.W. (1985) in DNA Cloning: A Practical

Approach (Vol. 1) (D.M. Glover, ed.), IRL Press Limited, Oxford, England, p. 49.

17. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.

18. Young, R.A. and Davis, R.W. (1983) Science 222, 778.19. Helfman, D.M., Feramisco, J.R., Fiddes, J.C., Thomas, G.P., and Hughes, S.H.

(1983) Proc. Natl. Acad. Sci. USA 80, 31.20. Grunstein, M. and Hogness, D. (1975) Proc. Natl. Acad. Sci. USA 72, 3961.21. Benton, W.D. and Davis, R.W. (1977) Science 196, 180.22. Vieira, J. and Messing, J. (1987) Methods Enzymol. 153, 3.23. Huang, M.T.F. and Gorman, C.M. (1990) Mol. Cell. Biol. 10, 1805.24. Murray, N.E., Brammar, W.J., and Murray, K. (1977) Mol. & Gen. Genet. 150, 53.25. Mierendorf, R.C., Percy, C., and Young, R.A. (1987) Methods Enzymol. 152, 458.26. Chomczynski, P. and Sacchi, N. (1987) Anal. Biochem. 162, 156.27. Simms, D. (1995) Focus 17, 39.28. McDonnell, M.W., Simon, M.N., and Studier, F.W. (1977) J. Mol. Biol. 110, 119.29. Gerard, G.F. and Miller, K. (1986) Focus 8:3, 5.30. Matathias, A.S. and Komro, C. (1989) Focus 11, 79.31. D’Alessio, J.M. and Noon, M.C. (1989) Focus 11, 49.

6

30

Product Size Cat. NoCombination SystemsSuperScript® Plasmid System and one set 19625-011 ElectroMAX™ DH10B™ Competent Cells

Products for Purification

Micro-FastTrack™ 2.0 mRNA Isolation Kit 20 reactions K1520-02TRIzol® Reagent 100 mL 15596-026 200 mL 15596-018TRIzol® LS Reagent 100 mL 10296-010 200 mL 10296-028UltraPure™ Guanidine Hydrochloride 500 g 15502-016Guanidine Isothiocyanate 500 g 15535-016UltraPure™ DEPC-Treated Water 4 × 1.25 mL 10813-012UltraPure™ Phenol 500 g 15509-037UltraPure™ Phenol:Chloroform:Isoamyl Alcohol, (25:24:1, v/v/v) 100 mL 15593-031 RNaseOUT™ Recombinant Ribonuclease Inhibitor 5000 units 10777-019RNase AWAY® Reagent 250 mL 10328-011

Products for TransformationMAX Efficiencyl® DH5α™ Competent Cells 1 mL 18258-012MAX Efficiencyl® DH10B™ Competent Cells 1 mL 18297-010IPTG 1 g 15529-019S.O.C. Medium 10 × 10 mL 15544-034X-gal 100 mg 15520-034

Products for ElectroporationElectroMAX™ DH10B™ Cells 5 × 0.1 mL 18290-015ElectroMAX™ DH12S™ Cells 5 × 0.1 mL 18312-017

Other Related ProductsSuperScript® II Reverse Transcriptase 10,000 units 18064-014Second-Strand Buffer 0.5 mL 10812-014Oligo(dT)12-18 Primer 25 µg 18418-012

Product Size Cat. NoUltraPure™ Glycogen 100 µL 10814-010cDNA Size Fractionation Columns 3 columns 18092-0151 Kb Pus DNA Ladder 250 µg 10787-018UltraPure™ Acrylamide 500 g 15512-023UltraPure™ 10 mg/mL Ethidium Bromide 10 mL 15585-011UltraPure™ 10X TAE Buffer 1 L 15558-042 4 L 15558-026 UltraPure™ 10X TBE Buffer 1 L 15581-044 10 L 15581-028UltraPure™ 1 M Tris-HCl (pH 7.5) 1 L 15567-027

Related Products

Headquarters5791 Van Allen Way | Carlsbad, CA 92008 USA | Phone +1 760 603 7200 | Toll Free in USA 800 955 6288For support visit lifetechnologies.com/support or email techsupport@lifetech.com

lifetechnologies.com18 January 2013