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InvitroScript™CAP KitVersion C
00061625-0044
InvitroScript™CAPSP6 In Vitro Transcription Kit
For the synthesis of capped mRNA trasnscripts from the SP6 promoter
Catalog no. K755-01
U.S. Headquarters: European Headquarters:Invitrogen Corporation Invitrogen BV1600 Faraday Avenue PO Box 2312, 9704 CH GroningenCarlsbad, CA 92008 The NetherlandsToll Free Tel: (800) 955-6288 Toll Free Tel: 00800 5345 5345Tel: (760) 603-7200 Toll Free Fax: 00800 7890 7890Fax: (760) 603-7201 Tel: +31 (0) 50 5299 299E-mail: [email protected] Fax: +31 (0) 50 5299 281Web: www.invitrogen.com E-mail: [email protected]
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Kit Contents and Storage
Shipping andStorage
The InvitroScript™CAP Kit is shipped on dry ice.Please store the kit at -20°C upon receipt. Do not store in a frost-free freezer. (Thewarming and cooling cycles of frost-free freezers are detrimental to enzymes.)
InvitroScript™CAPSP6 In VitroTranscription Kit
The kit contains enough reagents for twenty-five 20 µl reactions.
Reagent Composition Volume10X Enzyme Mix Placental RNase inhibitor
SP6 RNA polymerase(proprietary)
55 µl
10X Transcription Buffer (proprietary) 60 µl2X Ribonucleotide Mix 10 mM each of ATP, CTP, UTP 2 mM
GTP and 8 mM Cap Analog260 µl
20 mM GTP -- 2 x 30 µlGel Loading Buffer 80% formamide
0.1% xylene cyanol0.1% bromophenol blue2 mM EDTA
1 ml
RNase-free, deionized water -- 1 mlSinRep/lacZ template,linearized
0.5 µg/µl in DEPC-treated TE, pH 7.5 10 µl (5 µg)
ProductSpecifications
All components of the InvitroScript™CAP Kit are tested in an in vitro transcriptionreaction following the protocols described in the Sindbis Expression System manual. A20 µl reaction containing 1 µg of SinRep/lacZ template must yield at least 20 µg ofpolyadenylated RNA following a two hour incubation at 37°C. The RNA is analyzed bygel electrophoresis to ensure synthesis of the expected RNA product and the absence ofRNase in each kit component.
Technical Service For Technical Service, please call, write, fax or E-mail:U.S. Headquarters: European Headquarters:Invitrogen Corporation Invitrogen BV1600 Faraday Avenue PO Box 2312, 9704 CH GroningenCarlsbad, CA 92008 The NetherlandsToll Free Tel: (800) 955-6288 Toll Free Tel: 00800 5345 5345Tel: (760) 603-7200 Toll Free Fax: 00800 7890 7890Fax: (760) 603-7201 Tel: +31 (0) 50 5299 299E-mail: [email protected] Fax: +31 (0) 50 5299 281Web: www.invitrogen.com E-mail: [email protected]
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Table of Contents
Kit Contents and Storage ........................................................................................................... iii
Table of Contents ......................................................................................................................... v
Introduction.................................................................................................................................. 1Overview........................................................................................................................................................ 1
Methods......................................................................................................................................... 2FastStart ........................................................................................................................................................ 2Preparation of the DNA Template .............................................................................................................. 3In Vitro Transcription of DNA Templates .................................................................................................. 5Optimization of In Vitro Transcription....................................................................................................... 7Technical Assistance ..................................................................................................................................... 9
Appendix..................................................................................................................................... 11Analysis of RNA Quantity.......................................................................................................................... 11Miniprep Plasmid Preparation.................................................................................................................. 12Technical Service ........................................................................................................................................ 14References.................................................................................................................................................... 16
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1
Introduction
Overview
Description of Kit The InvitroScript™CAP SP6 In Vitro Transcription Kit enables synthesis of high yields ofin vitro transcribed capped RNA from the SP6 promoter. Most eukaryotic mRNAs have a7-methyl-guanosine cap structure at the 5´ end of the transcript. InvitroScript™CAPsynthesizes capped mRNAs by including the cap analog m7G(5´)ppp(5´)G in the SP6 invitro transcription reaction. The cap analog is only incorporated at the first or 5´ terminalG of the transcript. To make the InvitroScript™CAP Kit easy to use, all four ribonucleo-tides and the cap analog are provided in a single solution. The cap analog:GTP ratio is4:1, which is optimal for maximizing both the yield of RNA and the fraction of RNAswith a cap structure.To further maximize the yield of mRNA, reaction conditions have been optimized in thepresence of high nucleotide concentrations. The InvitroScript™CAP kit contains areaction buffer; a nucleotide mix containing the cap analog; additional GTP; an enzymemix containing SP6 polymerase, placental RNase inhibitor and other agents necessary forhigh yields; and the SinRep/lacZ control template for generating an 11.2 kb transcript.Each kit contains sufficient reagents for 25 transcription reactions (20 µl each). A trans-cription reaction using the control template supplied with the kit will yield 10-20 µg offull-length, capped RNA.
For MoreInformation
For more information on in vitro transcription, please refer to (Aziz and Soreq, 1990;Krieg, 1990; Krieg and Melton, 1987; Schenborn and Mierindorf, 1985).
ExperimentalOutline
The table below outlines the steps necessary to produce capped RNA transcripts using theInvitroScript™CAP Kit.
Step Action1 Isolate the DNA template using CsCl gradient centrifugation or the miniprep
procedure on page 12.2 Linearize 2-20 µg of the DNA template to generate transcripts of defined
size in the in vitro transcription reaction.3 Perform in vitro transcription using 1 µg of linearized DNA template.4 Use the transcription reaction directly to electroporate BHK cells if you are
using the Sindbis Expression System.ORPrecipitate and quantitate the RNA transcripts to use immediately, or storein aliquots at -80°C.
Important All reagents and equipment (e.g. buffers, gels, pipet tips, gel apparatus, etc.) thatcome in contact with RNA transcripts should be RNase-free.
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Methods
FastStart
Introduction The procedures below are designed to enable you to quickly get started with theInvitroScript™CAP kit. For more information on the in vitro transcription reaction, pleasesee pages 5-8.
RestrictionDigestion of theDNA Template
1. Purify template DNA by CsCl gradient centrifugation or the miniprep procedure onpage 12. For more information, please see pages 3 and 4.
2. Digest 2-20 µg of your DNA template with the appropriate restriction enzyme.3. Terminate the restriction digest with 1/20 volume of 0.5 M EDTA, pH 8.04. Phenol extract the restriction digest and ethanol precipitate the DNA with 1/10 volume
of RNase-free 5 M ammonium acetate, pH 7.4 and 2 volumes of 100% ethanol.5. Resuspend the DNA to a concentration of 0.5 µg/µl in RNase-free TE or water. You
will need 1 µg of linearized DNA for each in vitro transcription reaction.
In VitroTranscription
All reagents and equipment used in this section should be RNase-free.1. Thaw the 10X Transcription Buffer, the 2X Ribonucleotide Mix, and the RNase-
free water. Keep the 10X SP6 Enzyme Mix on ice.2. Set up the in vitro transcription reaction at room temperature in a microcentrifuge
tube by mixing the reagents in the following order:
RNase-free water 2 µlLinearized DNA template at 0.5 µg/µl (1 µg) 2 µl2X Ribonucleotide Mix 10 µl20 mM GTP* (optional) 2 µl10X Transcription Buffer 2 µl10X SP6 Enzyme Mix 2 µlFinal Volume 20 µl
*Inclusion of extra GTP is needed to ensure full-length transcript of the control template (seepage 6 and page 8)
3. Mix the reaction gently and incubate for 2 hours at 37°C.4. Before terminating the reaction, check the RNA by mixing 1 µl of the transcription
reaction with 3 µl of the gel loading dye, heating for 3-5 minutes at 80-90°C, andrunning on a 1% agarose gel. If the amount and quality of the RNA is sufficient, youmay terminate the reaction.
5. To terminate the in vitro transcription reaction, add 115 µl RNase-free water and15 µl of a 5 M ammonium acetate, 100 mM EDTA solution.
6. Extract the reaction once with phenol/chloroform and once with chloroform.7. Precipitate the RNA by adding 1 volume of isopropyl alcohol and mixing well. Chill
the reaction at -20°C for 15 minutes.8. Centrifuge at +4°C for 15 minutes at maximum speed to pellet the RNA. Remove
the supernatant and resuspend the RNA in RNase-free water or TE buffer. Quanti-tate the amount of RNA and use immediately or store at -80°C.
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Preparation of the DNA Template
Introduction Once you have your recombinant construct, you will need to prepare plasmid for in vitrotranscription. This requires preparing clean DNA and linearizing it prior to use in the invitro transcription reaction. Recommendations are provided below to ensure properpreparation of the DNA template.
General MolecularBiologyTechniques
For help with plasmid preparations and restriction enzyme digests, please see MolecularCloning: A Laboratory Manual (Sambrook et al., 1989) or Current Protocols inMolecular Biology (Ausubel et al., 1994).
ImportantThe plasmid DNA must be free of RNA and RNase before performing in vitrotranscription. RNA will inhibit transcription and may lower transfection efficiencies.RNase will degrade your transcripts.
PlasmidPreparation
You will need at least 1 µg of DNA template for each in vitro transcription reaction. Werecommend purifying your DNA using CsCl gradient centrifugation or using the plasmidminiprep procedure on page 12. This method uses RNase to eliminate RNA and aProteinase K digestion to remove the RNase. It is very important to eliminate RNase toavoid degradation of your transcripts. After isolation of plasmid DNA, be sure todetermine the concentration of your sample using UV absorbance, fluorescence, or theDNA DipStick™ Kit (Catalog no. K5632-01).
Before Starting theRestrictionDigestion
Because you will be generating RNA, it is very important that any solution that comes incontact with the RNA be made with diethylpyrocarbonate-treated (DEPC) water andautoclaved (Sambrook et al., 1989). All plasticware should be RNase-free. Be sure towear gloves. You will need to have available the following solutions and supplies:• Restriction enzymes and buffers• Phenol/chloroform (1:1)• 0.5 M EDTA, pH 8.0• RNase-free 5 M ammonium acetate, pH 7.4• 100% ethanol• RNase-free water or TE buffer
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Preparation of the DNA Template, continued
RestrictionDigestion
Digestion with a restriction endonuclease will generate templates of a defined size for usein the in vitro transcription reaction. You may make either sense or anti-sense transcripts,depending on the position of the SP6 promoter relative to the insert of interest. If usingthe Sindbis Expression System, the plasmid should be digested with a restriction enzymethat cleaves on the 3´ side of the insert (carboxy-terminal end with respect to proteincoding). Note: The SinRep/lacZ template is provided linearized. This template may beadded directly to the transcription reaction (page 5).1. Use 2-20 µg of your construct for restriction digestion. You will need 1 µg of
linearized DNA for each in vitro transcription reaction.2. Terminate the restriction digest with 1/20 volume of 0.5 M EDTA, pH 8.03. Phenol extract the restriction digest and ethanol precipitate the DNA with 1/10
volume of 5 M ammonium acetate, pH 7.4 and 2 volumes of 100% ethanol.4. Resuspend the DNA to a concentration of 0.5 µg/µl in RNase-free TE or water. You
may wish to check an aliquot of your linearized template on an agarose gel toanalyze quantity and quality.
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In Vitro Transcription of DNA Templates
Introduction At this point, you should have purified, linearized DNA template at a concentration of0.5 µg/µl. You are now ready to produce capped RNA transcripts using theInvitroScript™CAP SP6 In Vitro Transcription Kit.
SinRep/lacZControl Template
Linearized SinRep/lacZ template is included in the InvitroScript™CAP SP6 In VitroTranscription Kit as a transcription control. The 13104 bp DNA template is supplied as a0.5 µg/µl solution and can be used directly in the in vitro transcription reaction with nofurther preparation.This template may be used as an expression control in the Sindbis Expression System.Please refer to the Sindbis Expression System manual for more information.
Before Starting Prepare an RNase-free 5 M ammonium acetate, 100 mM EDTA solution if you wish toterminate the transcription reaction (see Step 6, next page). If you are using this kit inconjunction with the Sindbis Expression System, you do not need to terminate thereaction before proceeding to transfection.
In VitroTranscription
If you wish to quantitate the transcription reaction, refer to page 11 before starting thereaction. A typical reaction should yield 10-20 µg of RNA from 1 µg of linearizedtemplate.1. Thaw the 10X Transcription Buffer, the 2X Ribonucleotide Mix, and the RNase-
free water. Briefly vortex the 10X Transcription Buffer and the 2X RibonucleotideMix. Briefly centrifuge all reagents before opening to prevent loss of reagent orpossible contamination of material that may be present around the rim of the tube.Keep the 10X SP6 Enzyme Mix on ice.
2. Set up the in vitro transcription reaction at room temperature in a microcentrifugetube by mixing the reagents in the following order:
RNase-free water 2 µlLinearized DNA template at 0.5 µg/µl (1 µg) 2 µl2X Ribonucleotide Mix 10 µl20 mM GTP* (optional) 2 µl10X Transcription Buffer† 2 µl10X SP6 Enzyme Mix 2 µlFinal Volume 20 µl
*Inclusion of extra GTP is needed to ensure a full-length transcript of the controltemplate (see next page)
†Do not set up the reaction on ice as the 10X Transcription Buffer will precipitate.3. Mix the reaction gently and incubate for 2 hours at 37°C.
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In Vitro Transcription of DNA Templates, continued
In VitroTranscription,continued
4. Before terminating the reaction, check the quality of the RNA by mixing 1 µl of thetranscription reaction with 3 µl of the gel loading dye, heating for 3-5 minutes at80°C-90°C, and running on a 1% agarose gel.
Note: RNA run on the agarose gel can be visualized by staining with ethidiumbromide. Since this gel is nondenaturing, this procedure will only reflect the qualityand quantity of the RNA. A nondenaturing gel cannot be used to accuratelydetermine the size of the RNA. You may wish to compare the quality of the RNAband with DNA standards such as digested λDNA. The RNA band should bediscreet and relatively thick in comparison to the DNA bands. Please note that youmay see dimers of single-stranded RNA.
5. If the amount and quality of the RNA is sufficient, you may terminate the reaction.(If you are using this kit in conjunction with the Sindbis Expression System, you donot need to terminate the reaction before proceeding to transfection. Proceed totransfection of BHK cells or aliquot the RNA in 10 µl samples and freeze at -80°Cfor storage.)
6. To terminate the in vitro transcription reaction, add 115 µl RNase-free water and15 µl of the 5 M ammonium acetate, 100 mM EDTA solution.
7. Extract the reaction once with phenol/chloroform and once with chloroform.Transfer the aqueous solution to a new tube.
8. Precipitate the RNA by adding 1 volume of isopropyl alcohol and mixing well. Chillthe reaction at -20°C for 15 minutes. Precipitation with isopropyl alcohol reducesthe amount of free nucleotides precipitated with the RNA.
9. Centrifuge at +4°C for 15 minutes at maximum speed to pellet the RNA. Removethe supernatant and resuspend the RNA in RNase-free water or TE buffer.Quantitate the amount of RNA and use immediately or store at -80°C. If you aregoing to use the RNA immediately, keep the sample on ice.
ImportantSince the control template is greater than 10 kb, you must add 1-2 µl of 20 mM GTP inthe transcription reaction as the concentration of GTP will become limiting during thereaction. Adding GTP will decrease the fraction of transcripts containing the cap (since itdecreases the ratio of the cap analog to GTP), but will increase the yield of full-lengthproduct. If your template is greater than 5 kb, you may need to add additional GTP toensure full-length transcripts. Please see page 8 for recommendations on how much GTPto add.
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Optimization of In Vitro Transcription
Introduction The in vitro transcription reaction on page 5 has been designed to generate high yields ofRNA transcripts from longer templates such as those produced using the SindbisExpression System. This usually means adding additional GTP to the transcriptionreaction. If you wish to use the kit to transcribe templates smaller than 5 kb, it is notnecessary to add any additional GTP. Simply omit the 20 mM GTP solution from the listof reagents on page 5 and proceed as described. For templates shorter than 0.3 kb, theincubation time, template or polymerase concentration may need to be adjusted tomaximize reaction yields.
OptimizingTranscription ofShorter Templates
In general, as the template size decreases, the number of RNA copies of the template mustincrease to achieve the same mass yield (10-20 µg RNA). For example, if you use 1 µg ofa 5 kb plasmid to generate 20 µg of a 500 bp RNA transcript, the yield will be about 400copies per template molecule. Using the same plasmid and generating 20 µg of a 50 bptranscript will yield 4000 copies per template molecule. Since the template needs to becopied many more times to generate microgram amounts of the 50 bp transcript, theinitiation step of the transcription reaction becomes the rate-limiting step. There are threebasic parameters to optimize yields under conditions where transcription initiation is rate-limiting: incubation time, template concentration, and SP6 RNA polymeraseconcentration.Increase Incubation TimeThe incubation time may be increased which allows each SP6 RNA polymerase toperform a greater number of initiation events. This is the easiest variable to change andshould be tried first. Try increasing the incubation time to 4 or 6 hours.Adjust Template ConcentrationIt is important to remember that for short templates, an equal mass represents a largermolar amount of DNA when compared to longer templates. It is possible to saturate theSP6 RNA polymerase in the transcription reaction using shorter templates. Approximatelyone picomole of template (i.e. one picomole of promoter sequence) will saturate the SP6RNA polymerase. In practice, this means that 60 to 70 ng of a 100 bp DNA template willsaturate the polymerase. In general, for optimum yield of short transcripts, use about 0.5 to2 pmoles of template per reaction. This will ensure that the polymerase will always bebound to template.Note: It may be necessary to concentrate the DNA template in order to be able to add thedesired amount in a reasonably small volume.Increase SP6 Polymerase ConcentrationIn reactions using much smaller templates, increasing the amount of SP6 RNAPolymerase may increase yields. Add 200 units of pure, high concentration SP6 RNApolymerase (not the 10X Enzyme Mix) to the in vitro transcription reaction and test forincreased yield.
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Optimization of In Vitro Transcription, continued
OptimizingTranscription ofLonger Templates
When synthesizing transcripts greater than 5 or 6 kb, GTP levels in the reaction willbecome rate limiting and may result in lower yields, a high proportion of less than full-length product, or both. Extra GTP is supplied in the kit to supplement the transcriptionreaction when using longer templates.Adding Extra GTPNormally the ratio of cap analog to GTP in the 2X Ribonucleotide Mix is 4:1. This ratiois decreased with the addition of 20 mM GTP. As more GTP is added, the amount of full-length transcripts will increase, but the fraction of transcripts that are capped willdecrease. We recommend using the minimum amount of GTP to increase the yield of full-length transcripts. The table below shows the effect of additional GTP on the fraction ofRNA transcripts capped.
Added GTP Final Cap Analog:GTP Ratio Fraction of Transcripts Capped0 µl 4.0:1 80%1 µl 2.0:1 67%2 µl 1.3:1 57%3 µl 1.0:1 50%
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Technical Assistance
Troubleshooting Use the tables below to troubleshoot the in vitro transcription reaction.
If the... Then,...yield from your invitro transcriptionreaction is low,
perform the in vitro transcription reaction using the controltemplate. You should obtain 10-20 µg of RNA. Proceed to thenext table.
If the... Then,...yield from thecontrol reaction islow,
verify the yield by a second method. You may quantitate yieldusing the radioactive method on page 11 or you may run analiquot out on a gel to estimate yield.
If the.... Then, ....yield of RNA as determined bytwo methods is still low,
there may be a technicalproblem with the way the kit isbeing used (e.g. the order ofaddition of reagents isimportant as spermidine in theTranscription Buffer mayprecipitate the DNA template ifadded to the reaction first)Review the in vitrotranscription procedure.
yield from thecontrol reaction isbetween 10-20 µg,
repeat the in vitro transcription reaction using the controltemplate and a mixture of the control template and your template.This will indicate whether there are inhibitors of the transcriptionreaction in your template solution.
If the... Then, ...control reaction is inhibitedby the presence of yourtemplate,
the transcription reaction isinhibited by something in yourDNA template solution. Refer toEliminating Inhibitors in theTemplate Solution, next page,for possible solutions.
control reaction is notinhibited by the presence ofyour template,
there is something inherent toyour template that results in lowyields. You may extend theincubation time to 4 or 6 hours toimprove yields.
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Technical Assistance, continued
EliminatingInhibitors in theTemplate Solution
Use the following procedures to eliminate inhibitors from your DNA template.To eliminate RNase, SDS, or NaCl from your template:1. Treat DNA with Proteinase K (100-200 µg/ml) and SDS (0.5%) for 30 minutes at
50°C.2. Extract with phenol/chloroform and dilute the template several-fold before ethanol
precipitation.3. Wash the template pellet thoroughly with 70% ethanol.If this treatment fails to eliminate inhibitors, purify DNA by CsCl gradient centrifugation,making sure to remove all of the CsCl by dialysis or several rounds of ethanolprecipitation.
Size of RNATranscript
The table below provides suggestions to troubleshoot problems with the size of yourRNA transcript.
Problem Probable Cause SolutionSize of the RNA productis not what is expected
RNA transcript hassecondary structure thatcauses it to migrateanomalously on a nativeagarose gel
Run the RNA on adenaturing gel to determinethe correct size.
Denaturing gelelectrophoresis shows thepresence of multiple bandsor a single band of lessthan the expected size.
Premature termination bythe SP6 polymerase
Decrease the reactiontemperature to 30°C, 20°C,or 10°C.
Technical Service For Technical Service, please call, write, fax or E-mail:U.S. Headquarters: European Headquarters:Invitrogen Corporation Invitrogen BV1600 Faraday Avenue PO Box 2312, 9704 CH GroningenCarlsbad, CA 92008 The NetherlandsToll Free Tel: (800) 955-6288 Toll Free Tel: 00800 5345 5345Tel: (760) 603-7200 Toll Free Fax: 00800 7890 7890Fax: (760) 603-7201 Tel: +31 (0) 50 5299 299E-mail: [email protected] Fax: +31 (0) 50 5299 281Web: www.invitrogen.com E-mail: [email protected]
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Appendix
Analysis of RNA Quantity
Purpose The purpose of the following procedure is to determine the quantity of RNA producedusing the InvitroScript™CAP SP6 In Vitro Transcription Kit (page 5).
Before Starting You will need to prepare or have on hand the following reagents and supplies. Allreagents and equipment needs to be RNase-free.• [α-32P] UTP, 10 µCi/µl (The concentration of GTP limits the amount of RNA that can
be synthesized; therefore, it is important to use a tracer other than GTP.)• G-50 Sephadex spin columns (Pharmacia)• 37°C heat block or water bath• Whatman 3MM paper• Scintillation cocktail
Procedure 1. After setting up the in vitro transcription reaction (page 5, Step 2), transfer 5 µl ofthe reaction to a new tube and add 0.5 µl [α-32P] UTP.
2. Incubate for 2 hours at 37°C.3. Add RNase-free, deionized water to 100 µl to stop the reaction.4. Remove 50 µl of the sample and centrifuge through a G-50 spin column according
to manufacturer's instructions. This will remove unincorporated nucleotides. Flow-through will contain mRNA (incorporated [α-32P] UTP). Reserve the remainingsample (50 µl) to determine the total (incorporated + unincorporated) amount ofradioactivity in the transcription reaction.
5. Take 5 µl aliquots from the two RNA samples in Step 4, spot on individuallylabeled Whatman 3MM paper disks, and allow to dry.
6. Add the papers to scintillation cocktail and count in a scintillation counter. The ratioof the incorporated to total radioactivity from Step 4 represents the fraction ofradiolabeled nucleotide incorporated into RNA.
Calculation ofYield
Incorporation of 1% of the 32P counts corresponds to 1.3 µg of RNA synthesized usingthe InvitroScript™CAP SP6 In Vitro Transcription Kit. Use the counts determined inSteps 1-5, above, to calculate the yield of mRNA.Calculation of yield:
Incorporated [α-32P] UTP x 100 x 1.3 µg RNA = µg RNA per 20 µl reaction
Total [α-32P] UTP
SinRep/lacZ (Incorporated) 150079 cpmSinRep/lacZ (Total) 890989 cpm
(150079/890989) (100) (1.3 µg) = 21.9 µg/20 µl reaction
Note that this gives the yield in a 20 µl reaction; therefore if the reaction volume isdoubled so is the calculated yield. The theoretical yield of a 20 µl reaction is 40 µg.
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Miniprep Plasmid Preparation
Introduction Generally, the cleaner the template DNA, the greater the yield from the in vitrotranscription reaction. Template DNA purified on a CsCl gradient gives the highestyields. However, the miniprep procedure below yields DNA template of sufficient qualityfor in vitro transcription. The procedure uses a Proteinase K digestion step to ensurethat all ribonuclease is degraded prior to the in vitro transcription reaction.
Before Starting You will need the following reagents and equipment:• ~1.5 ml of bacterial culture (E. coli transformed with your construct)• Sterile 50 mM glucose, 10 mM EDTA, pH 8, 25 mM Tris-HCl, pH 8.0• 0.2 N NaOH, 1% SDS (make fresh)• 3 M potassium/5 M acetate (for 100 ml, mix 60 ml 5 M potassium acetate and 11.5 ml
glacial acetic acid with 28.5 ml deionized water)• 100% Ethanol• 10 mg/ml RNase• TE with 20 µg/ml RNase (Add 1 µl of 10 mg/ml RNase to 500 µl TE)• Appropriate restriction enzymes and buffers• 20 mg/ml Proteinase K• Phenol/chloroform• 5 M ammonium acetate (RNase-free)• RNase-free water
Isolation of NucleicAcid and RNaseDigestion
The procedure below starts with the basic alkaline miniprep procedure to isolate nucleicacid.1. Centrifuge 1.5 ml of the bacterial culture at maximum speed in a microcentrifuge for
30 seconds at room temperature. Remove all the medium.2. Add 110 µl of 50 mM glucose, 10 mM EDTA, pH 8, 25 mM Tris-HCl, pH 8.0 to
the pellet and vortex vigorously to fully resuspend the bacterial pellet.3. Add 220 µl of 0.2 N NaOH, 1% SDS, invert the tube 10 times to mix, and incubate
on ice for 1 minute (or longer).4. Add 165 µl 3 M potassium/5 M acetate, vortex for 10 seconds, and incubate for
5 minutes on ice.5. Centrifuge at +4°C for 5 minutes at maximum speed in a microcentrifuge.6. Transfer the supernatant to a tube containing 1 ml ethanol, invert several times to
mix, and incubate on ice for 5 minutes.7. Centrifuge at +4°C for 5 minutes at maximum speed. Decant supernatant, centrifuge
briefly, and aspirate off residual supernatant.8. Resuspend nucleic acid pellet in 50 µl TE buffer containing 20 µg/ml RNase.
Vortex vigorously and incubate for 5 minutes at 37-42°C. Make sure pellet issolubilized.
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Miniprep Plasmid Preparation, continued
Restriction-Proteinase KDigestion
After removing RNA, the plasmid is ready to be linearized prior to in vitro transcription.Choose a restriction enzyme that will linearize the plasmid for sense or anti-sensetranscripts. After restriction digestion, Proteinase K is added to remove all enzymes. Thedigest is then extracted with phenol/chloroform and the DNA ethanol precipitated. Thevolume of the restriction digest should be at least 2-3 times the volume of the miniprepused. For example, if your miniprep volume is 10 µl, perform the restriction digest in a20-30 µl volume.1. Digest miniprep DNA (all or part) with the appropriate restriction enzyme. Digest at
37°C for at least 1 hour, using 0.3-0.8 units of restriction enzyme per microliter ofminiprep DNA.
2. Add a 1/10 volume of Proteinase K (20 mg/ml), mix by pipetting, and incubate at50°C for at least 30 minutes.
3. Extract the digest once with phenol/chloroform and remove the top, aqueous phaseto a new tube.
4. Add 1/10 volume of 5 M ammonium acetate (RNase-free) and 2 volumes of ethanol.Incubate at -20°C for at least 15 minutes.
5. Centrifuge the solution at maximum speed in a microcentrifuge for 15 minutes at+4°C. Decant supernatant, centrifuge briefly, and remove all the ethanol.
6. Resuspend DNA pellet in 10-20 µl RNase-free water per 50 µl of miniprep nucleicacid per 1.5 ml bacterial culture.
7. Determine concentration of the DNA. You will need 0.5-1.0 µg of template DNAper a 20 µl in vitro transcription reaction. This will be about 1-3 µl of the 10-20 µlpreparation in Step 6.
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Technical Service
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Technical Service, continued
Limited Warranty Invitrogen is committed to providing our customers with high-quality goods and services. Our goalis to ensure that every customer is 100% satisfied with our products and our service. If you shouldhave any questions or concerns about an Invitrogen product or service, please contact ourTechnical Service Representatives at:1-800-955-6288, extension 2 (US and Canada)00800 5345 5345 (toll free) or +31 (0) 50 5299 299 (Europe, Middle East, and Africa)1-760-603-7200 extension 2 (all others)Invitrogen warrants that all of its products will perform according to the specifications stated on thecertificate of analysis. The company will replace, free of charge, any product that does not meetthose specifications. This warranty limits Invitrogen Corporation’s liability only to the cost of theproduct. No warranty is granted for products beyond their listed expiration date. No warranty isapplicable unless all product components are stored in accordance with instructions. Invitrogenreserves the right to select the method(s) used to analyze a product unless Invitrogen agrees to aspecified method in writing prior to acceptance of the order.Invitrogen makes every effort to ensure the accuracy of its publications, but realizes that theoccasional typographical or other error is inevitable. Therefore Invitrogen makes no warranty ofany kind regarding the contents of any publications or documentation. If you discover an error inany of our publications, please report it to our Technical Service Representatives.Invitrogen assumes no responsibility or liability for any special, incidental, indirect orconsequential loss or damage whatsoever. The above limited warranty is sole and exclusive.No other warranty is made, whether expressed or implied, including any warranty ofmerchantability or fitness for a particular purpose.
16
References
Citations Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A.,and Struhl, K. (1994). Current Protocols in Molecular Biology (New York: GreenePublishing Associates and Wiley-Interscience).
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