Recombinant DNA Lab Practices

Christine Kelly12/4/2014BIOL3110LFall Semester, 2014

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Introduction:

Recombinant DNA technology is used in many labs today. It is used to clone certain

DNA strands with wanted genes. Recombinant DNA is DNA that has been brought together

through the use of restriction enzymes and bacteria hosts. The simplest definition of

recombinant DNA “is the process of making DNA molecules, created by joining DNA from two

different sources done in vitro” (Cain, Wenzel, & Walker, 2003). In this lab we learned the

process of created and growing bacteria that contained the recombinant DNA we created.

There is an importance to learning how to properly perform these lab procedures.

Recombinant DNA is important in many areas of study. Recombinant DNA studies have led to

advances and “astonishing improvements in medical diagnosis, prognoses, and therapies” (Berg

& Mertz, 2010). They have also led to development in many diverse fields such as “…chemistry,

evolutionary biology, paleontology, anthropology, linguistics, psychology, medicine, plant

science, and even forensics.” (Berg & Mertz, 2010) Just the diverse amount of fields that use

recombinant DNA technology is proof enough that knowing the processes and procedures are

important.

The procedures of recombinant DNA technology are described in the laboratory manual

provided to the class. There are three basic elements needed first in creating the recombinant

DNA. The basic elements are the desired DNA isolated from a source, a vector to carry the

DNA, and a host cell that will accept the vector and express the desired DNA. Once the basic

elements are gathered then “you must ‘cut’ a restriction fragment that contains the gene you

desire to close” (Cain, Wenzel, & Walker, 2003). These cuts should match with the same sticky

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ends so that complimentary sticky ends can form a recombinant plasmid .This was attempted in

the experiment.

In this experiment, Escherichia coli (E.coli), was used as both host bacteria and the

carrier of the plasmids to be obtained and made into recombinant DNA. The experiment was

meant to show how to purify, recombine and clone. The experiment used E. coli MM294

(pAMP) and E. coli MM294 (pKAN) to form a recombinant plasmid that contained the

resistance genes for ampicillin and kanamycin. This was done through purification of the

plasmids contains the wanted genes first. Then the two plasmids containing the genes of interests

are ligated to form the recombinant plasmid that was to be obtained. The bacteria hosts were then

made competent and the recombinant plasmids inserted into them so they could be plated for

growth and cloning. Hopefully this will demonstrate the process and procedures in created and

cloning recombinant DNA plasmids.

By demonstrating this procedure the question of how recombinant DNA is formed and

cloned should be answered. In answering this question the class will learn the importance of

recombinant DNA as well as the procedures in creating recombinant DNA. In knowing these

procedures one will be able to continue onto a career in a varied amount of fields and repeat the

procedures in much more detailed and needed research.

Methods and Materials:

First plasmid purification was performed. 50 ml of E.coli MM294 (pAMP) and E.coli

MM294 (pKAN) was obtained. Each sample was then purified using the PureYield Plasmid

Midiprep System. Once purified the samples were then tested for purity using a

spectrophotometer.

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Second restriction enzyme digestion of each sample was performed. A .2ug/ul dilution of

purified pAMP and a .2ug/ul dilution of purified pKAN were made. A single and a double digest

was performed on each sample. For the pAMP single digest, 8.5 ul of dH2O, 2ul of 10x buffer E,

2ul of BamHI and 7.5ul of pAMP were obtained. For the double digest of pAMP 6.5 ul dH2O,

2ul 10x buffer E, 2ul BamHI, 2ul HindIII, and 7.5 ul of pAMP were obtained. For the single

digest of pKAN, 8.5 ul of dH2O, 2ul 10x buffer E, 2ul of BamHI, and 7.5 ul of pKAN was

obtained. The double digest of pKAN contained 6.5ul dH2O, 2ul 10x buffer E, 2ul BamHI, 2ul

HindIII, and 7.5ul of pKAN were obtained. Each sample is then bump spun, flicked to mix and

then pulse spun. They are then incubated at 37 C for one hour.

Next Gel Electrophoresis was run on the samples. A .8% agarose gel was prepared in 1X

TAE. Then 5ul was taken from each tube from the previous step. 1ul of loading dye was added.

The gel was run for one hour at 90v-100v after each sample was loaded into a loading well as

well as a Lambda/HindIII molecular weight marker.

The next procedure is ligation of pAMP and pKAN. First samples of the pKAN and

pAMP digests from the previous steps were cleaned up using MinElute reaction cleanup kits.

Once cleaned up the digested pKAN and digested pAMP was incubated at 65 C for ten minutes.

Next a ligation tube was prepared. In the ligation tube 4.5ul of the double digested pAMP, 45 ul

of the double digested pKAN, 3ul 10x ligation buffer, 16 ul of dH2O, and 2ul of T4-ligase. The

tubes were then incubated at room temperature for 2-24 hours. Once incubated gel

electrophoresis is run on the samples.

Transformation of E.coli with the recombinant pAMPKan is the next procedure

performed. First 5ml of LB broth and E.coli MM294 is incubated over night at 37 C in a culture

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tube. In a 250ml flask add 50ml of LB broth and 1ml of E.coli MM294 from the overnight

culture then incubate at 37 C with vigorous shaking for two hours. Transfer 10 ml of E. coli

MM294 to a sterile 15 ml culture tube. Centrifuge at 3000 rpm for 10 minutes in a refrigerated

centrifuge. Then discard supernatant. Add 5ml cold sterile 50mM CaCl2 and gently suspend

pellet. Place cells on ice for 20 minutes. After incubation on ice for 20 minutes, centrifuge for

five minutes and then suspend the cells in 1ml CaCl2 after discarding the supernatant. Store LB

Broth with E. coli MM294 on ice for thirty minutes. With the .2ug/ul pAMP and the .2 ug/ul

pKAN from earlier make a new dilution of .005 ug/ul of each. In a 1.5 ml microtube put 200 ul

of the concentrated E.coli MM294 cells and 10 ul of the plasmid mixture. Make on each of the

following: +Lig incubated pAMP/KAN, + Lig non-incubated pAMP/KA, +pAMP, and +pKAN.

Place the four tubes on ice for 20 minutes. Heat shock after ice incubation for 90 seconds at 47

C. Then add 800ul of LB broth to each tube. Incubate again at 37 C for 40 minutes. Spread 100

ul for the tubes on plates with the following, LB, LB/AMP, LB/KAN, and LB/AMP+KAN.

Incubate plates over night at 37 C. Then the results are tested by inoculating two colonies, one

from the LB/Amp plate and one from the LB/KAN plate. The DNA is again purified using the

PureYield Miniprep System.

Flow Chart:

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Results:

Purification results:

A260 A280 A320

pAMP 1.158 .649 .052

pKAN .463 .422 .363

Table 1

pKAN = (.463-.363)/(.422-.363) = 1.7

pAMP = 1.158/.649 = 1.8

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Restriction Enzyme Digestion:

Figure 1 Gel Electrophoresis of the digestion of pKAN and pAMP. Done at 97v for 1 hour on 10/9/14. Lanes listed from bottom to top. Lane 1 – Lambda/HindIII marker. Lane 2 – pAMP undisgested, Lane 3- pAMP single digestion, Lane 4 – pAMP single digestion, Lane 5 –pAMP Double digestion, Lane 6 – pAMP double digestion, Lane 7- pKAN undigested. Lane 8 – pKAN single digest, Lane 9 – pKAN single digest, Lane 10 – pKAN double digest, Lane 11 –pKAN doublre digest, Lane 12 – Lambda/HindIII marker.

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In lane one, the lane on the bottom the Lambda/HindIII markers are present. The bands

are from the left to the right. 23130 bp, 9416 bp, 6557 bp, 4361 bp, 2322 bp, 2027bp, and the

band on the far right is at 564bp. In Lane 2 if the undigested pAMP bands. In lane 3 and lane 4

there is one band present at approximately 4500bp. In lane 5 and lane 6 there are two bands

present The one on the left is at approximately 4000 bp and the one on the right is near 750 bp.

In lane 7 is the undigested pKAN. In land 8 and lane 9 there is one band present at 4200 bp. In

lane 10 and lane 11 there are two bands present. One band is at the 3000bp position and the

second band is at the 2500bp position.

Ligation of pAMP and pKAN:

Figure 2 Gel electrophoresis of the ligation of pAMP and pKAN. Lanes are listed from left to right. Lane 1 – Lamda/HIndIII marker. Lane 2 – nonincubated ligation. Lane 3 – incubated ligation, Lane 4 – Lambda/HindIII marker.

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There are three bands present in lane three, which is the lane with the incubated ligation

of pAMP. The top band is at approximately 16000 bp. The second band is at approximately 6000

bp. The third band rests at around 4100 bp.

Transformation of E.coli mm294 with recombinant pAMP/KAN:

Plate Number of Colonies

+pLig Incubated +pLig no inc. +pAMP +pKAN

LB TNTC TNTC TNTC TNTC

LB/AMP 284 TNTC 167 0

LB/KAN 0 0 0 0

LB/AMP +

KAN

0 0 0 0

Table 2 Colony Counts

Figure 3 – Growth plates of transformed E.coli MM294

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Figure 4 Growth plates of transformed E. Coli MM294

Figure 5 Growth Plates of transformed E. Coli MM294

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Figure 6 Growth Plates of transformed E. Coli MM294

Purification of Transformed E.Coli MM294 :

A260 A280 A320

p/KAN .050 .045 .026

p/AMP .084 .063 .027

Table 3

Calculations:

p/KAN: (.050-.026)/(.045-.026) = 1.3

p/AMP: (.084-.027)/(.063-.027) = 1.6

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Figure 7 Final Gel of ligated enzymes. Run at 98v for 1 hour. Lanes are numbered from left to right. Lane 1 Lambda/HindII marker. Lane 2 LB/Amp. Lane 3 LB/Amp. Lane 4 LB/Kan. Lane 5 LB/KAN. Lane 6 pAMP. Lane 7 pAMP. Lane 8 LB/Amp digested. Lane 9 LB/Amp digested. Lane 10 LB/Kan digested. Lane 11 LB/Kan digested. Lane 12 pAMP + pKAN digested. Lane 13 pAMP + pKAN digested. Lane 14pAMp + pKAN.

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Lane Band SizeLambda/HindIII Marker 23130 bp,9416 bp, 6557 bp, 4361 bp, 2322 bp,

2027 bp. 564 bpLB/Amp No bandsLB/Amp No bandsLB/Kan No bandsLB/Kan No bandspAMP 2100bp, 9400 bp, 6600 bp, 4400 bp, 4000 bp,

2200 bppAMP 2100bp, 9400 bp, 6600 bp, 4400 bp, 4000 bp,

2200 bpLB/Amp Digest No bandsLB/Amp Digest No bandsLB/Kan Digest No bandsLB/Kan Digest No bands

pAMP + pKAN digest 700 bp, 1800 bp, 2100 bp, 3800 bppAMP+pKAN digest 700 bp, 1800 bp, 2100 bp, 3800 bp

pAMp + pKAN Ligation 9500 bp. 9300bp, 6100 bp, 4250 bp, 3800 bp, 2400 bp

Table 4

Discussion:

The first thing that was done was the E.coli MM294 (pAMP) and E.coli MM294 (pKAN)

was purified. In our results it can be seen that according the spectrophotometer the purified

pAMP and pKAN both fell within the range of 1.7-1.9 (table 1) indicating that the samples fall

within the purification standards. This means that the samples used were pure enough to yield the

genes wanted. This was followed by a restriction enzyme digest of the samples obtained.

When viewing figure 1 we can see the bands that represent the cuts of the plasmids by the

enzymes BamHI and HindIII. In lane two where the undigested pAMP is present there should

have been only one band at or near 4539 bp. Instead we can see many bands are visible in the

undigested pAMP lanes. This can be for many reasons. One reason is the handling of the DNA

during lyses, it could have been over incubated. Another reason is the plasma DNA may have

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unwound making it run slower. A third reason could be chromosomal contamination from over

mixing. The figure 8 will show the plasmid map for pAMP and pKAN.

In the following lanes figure 8 is used for the discussion of the results. In the two lanes

containing the single digest of pAMP, lanes 3 and 4 in figure 1, we can see a band located near

4500bp. This band is where it should be. The pAMP plasmid if 4539 bp long, and being cut by a

single enzyme it would become linear creating the band that we see present. In lane 5 and lane 6

of figure 1 we can see the two bands that are present. One band is at or near 4000 bp. This band

would closely represent the 3755 bp segment seen in figure 8 that contains the ampicillin gene.

The second band rests at or near 750 bp. This puts this band very close to the 784 bp that is

expected in a double digest of pAMP.

Lane 7 is undigested pKAN, here we can see the same occurrence as in the undigested

pAMP. There are many bands present which indicates that there is some kind of contamination.

It could be chromosomal DNA contamination or denaturing of the plasmid during lyses from

over incubation.

Lane 8 and Lane 9 are the single digest of pKAN plasmids. The band that is seen is close

to the expected 4207 bp length being at 4200bp. Lane 10 and lane 11 are the double digests of

pKAN. The first band present is near 3000 bp. The cut should have been closer to 2332 bp. The

second band is located near 2500 bp. This band should have been closer to1875 bp. Perhaps if

the gel had been run a little longer we may have reached the desired results. The bands in the

pKAN lanes are also very faint and harder to see. This could indicate degraded DNA.

Next ligation of pAMP and pKAN was performed. A gel was run to determine if ligation

had taken place. Figure 2 is and image of this gel. In figure 2 look at the third band, it is the one

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labeled incubated pAMP/pKAN, it shows that there are three bands. One band is located at

16000 bp. This band could be a concatamer, meaning it is many pieces annealed together. The

second band is located at 6000 bp. This band is very close to our desired 5830 bp band. This

shows us the results that we were hoping to see. This shows us that ligation has occurred

between the pAMP and pKAN fragments forming a recombinant plasmid as illustrated in the

figure below.

Figure 8 (Recombination of Antibiotic Resistent Genes)

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The final band rests at 4100 bp. This band is close to two possibilities. One it is very close to the

size of undigested pAMP. The other being it is close to size of the 3755 bp larger pAMP

fragment.

In table 2 and figures 3-6 the growth of E.coli MM294 with the ligated plasma of

pAMP/KAN was measured and observed. As it can be seen, in the plates with no antibiotics

present growth was rampant and there were too many colonies to count. In the plates with the

ampicillin antibiotic, labeled LB/AMP there was growth in all the plates except the plates spread

with E. coli MM294 (pKAN). This is to be expected. This is because the bacteria present did not

have the plasmid with the ampicillin resistant gene. There was no growth in the plates that had

kanamycin antibiotic in them. This was to be expected in the plates spread with E. coli MM294

(pAMP) since they did not contain the kanamycin resistant gene. All of the other plates should

have had some growth though. There are a few reasons we may not have had growth. One reason

could be because the ligation was not incubated long enough. Due to time constraints within the

classroom setting we only incubated for two hours. Incubation could take as long as 24 hours.

We also may not have made the cells competent enough. We only stored them on ice for 30

minutes when it could take up to two hours. For these reasons we also had no growth on the

plates that had both ampicillin and kanamycin antibiotics present. We would have needed fully

transformed E. Coli MM294 (pAMP/Kan), this would have allowed growth on the plate spread

with the ligation transformed E.coliMM294.

Next to confirm that we had the recombinant DNA we purified the transformed E. coli

MM294. In table 3 it can be seen that the purification yields were not within the range expected.

The pAMP purification numbers were very close. Next gel electrophoresis was run on the

purified samples and compared with samples from before. As seen in figure 7 and table 4 there

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were no bands present in the undigested or digested LB/Amp and LB/Kan tests. In figure 1 we

saw the lines in the pKAN lanes were very faint. This leads us to believe the DNA may be low

quality or degraded. The more the DNA was worked with through purification, ligation and then

purification again it seems the DNA was degraded to the point that it was no longer present. In

lanes 6 and 7 we had the same issue as we had before with the undigested pAMP. There are too

many bands present indicating chromosomal DNA contaminations or denaturation during the

lyses process. In Lane 14 that contains the pAMP+pKAN ligation there are several bands

present. The 9500 bp and the 9300 bp bands could be concatamers again. The 6100 bp band

present is close to the band we expect in a ligation of pAMP and pKAN fragments. The 4250 bp

band is close to the ligation of two pKAN fragments, meaning during the process they annealed

back together. The 3800 bp fragment is close to the annealing of two 1875 pKAN fragments and

the 2400 bp band is close to the annealing of a 784 bp pAMP fragment and an 1875 bp pKAN

fragment.

In the result obtained the desired product was not reached. This could be due to things

such a low concentration of pKAN DNA. If you look at figure 1 you can see the bands present

for the lanes with pKAN DNA are very faint and light. This could mean the reason why we did

not receive our desired results is the fact that we had such low concentrations to start with. Even

though the desired results were not reached, the practices and procedures of obtaining

recombinant DNA were learned. Through the process of first purifying to obtain the wanted

DNA plasmids and then through the ligation of the plasmids it was realized how important such

technology can be. The ability to transform host cells to carry genes that are desired can lead to

many new discoveries.

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Works CitedBerg, P., & Mertz, J. E. (2010, January). Personal Reflections on the Origins and Emergence of

Recombinant DNA Technology. Genetics, 9-17.

Cain, G. D., Wenzel, D. C., & Walker, J. R. (2003). BIOL 3110L Labroatory Manual. University of Georgia.

Recombination of Antibiotic Resistent Genes. (n.d.). Retrieved 11 28, 2014, from DNA Learning Center Lab Protocols: http://labprotocols.dnalc.org/files/028_recombination_of_antibiotic_resistance_genes.pdf