While our transformations were incubating, we obtained 6 petri dishes. Four of the plates were LB/kanamycin/IPTG plates (labeled plates 1-4), one was a LB/kanamycin plate (labeled as K2 and also our negative control #1) and the last one was just a LB plate with no kanamycin or IPTG (labeled as K4). After 45 minutes of incubation, we began plating the cells onto each plate. 80 ul from each of ligations were plated onto their respected plates (1-4). Additionally, 20 ul of Ligation #2 was plated onto K2 and 20 ul of Ligation #4 was plated onto K4. We then placed each inverted plate in a 37°C incubator and allowed them to grow overnight.
Miniprep of Plasmid DNA
The purpose of this experiment was to isolate plasmids from 3 different liquid cultures of E. coli
We were given three 3ml cultures of E.coli that were picked from the petri dishes we plated previously. The first culture contained a colony that fluoresced green under UV light and was the second contained a colony that did not fluoresce green under UV light. Both cultures were picked from one of our experimental ligations that were plated on our LB/Kan/IPTG plates. The third colony was picked from ligation #2 that was plated on the LB/Kan plate and was considered our “unknown sample”.
For our miniprep experiment, we began by transferring 1.2 ml of each of our cultures into separate microcentrifuge tubes. We then pelleted the cells by centrifuging our tubes at max. speed for one minute and removed the supernatant with a P-1000 micropipette. Next we added 200 ul of Cell Suspension Solution to our tubes and vortexed each tube to disperse the cells in the solution. We then mixed in 200 ul of Cell Lysis Solution to each of our tubes and then added 200 ul of Neutralization Solution to create a precipitate. To pellet the precipitate we centrifuged our solutions for 5 minutes, then extracted the supernatant which would contain our DNA very carefully. For each of our minipreps, we prepared one Wizard Minicolumn by removing the plunger of a 3ml syringe and attaching the syringe barrel to the Luer-Lok ® extension of our labeled minicolumns. Next we were given 1 ml tubes of resuspended DNA Purification Resin to mix into each of our preps where we then transferred 1 ml of the resin suspension to the barrel of the syringe/minicolumn. To load the plasmid DNA into the barrel, we pipetted the supernatant out and transferred it into the barrel of the syringe containing the purification resin. We then pushed the DNA/resin mixture into the minicolum, then disassembled our syringe/minicolumn properly before pipetting 2.0 ml of Column Wash Solution and pushing it through the resin of the minicolumn. Lastly, we dried the resin by transferring it onto a 1.5 ml microcentrifuge and centrifuging it for 2 minutes before finally transferring the solution into a final tube. After 50 ul of TE buffer was added, we centrifuged our tubes for 20 seconds where they were then ready to be used directly as plasmid DNA.
Restriction Digest of Plasmid DNA
The purpose of this experiment was to run a variety of restriction digests (or none at all) through our isolated plasmids in order to determine the presence or absence of inserted plasmids for each of our clones.
We set up a total of two digests for each of our three plasmid DNAs isolated earlier from our miniprep, one with NotI/NcoI enzymes and one with just the NcoI enyme for a complete total of six restriction digests. We were then given two“ master mixes” containing the correct volumes of the reagents needed for either a single digests or a double digests and pipetted out 15 ml of each master mix as well as 5 ml of plasmid DNA into their appropriate tubes to get a total of 20 ul for each of our digests. While incubating our digest at 37 °C for 1 hour, we prepared our “undigested” samples for each plasmid (3 total). Each undigested sample contained all the same ingredients at the same concentration as the digested samples except no enzyme was added. This was all also given to us (besides the DNA) in a different master mix. We then pipetted out 15 ml of the undigested master mix into three microcentrifuge tubes and added 5 ml of DNA.
After being incubated at -20°C for one week, we ran our 9 digest samples (3 single digests, 3 double digests and 3 undigested) through a 50 ml 0.9% agarose gel at 60 V initially before turning it up to 110 V to allow the DNA to travel faster.
Polymerase Chain Reaction (PCR)
The purpose of this experiment was to properly set up and run polymerase chain reactions for our recombinant plasmids using specified primers to confirm the identity of our plasmids.
The primers we used, pAD1sense and pAD1anti were specific for our pET41-EGFP recombinant plasmids. The DNA templates we used were the three plasmids we isolated earlier in our miniprep, a given positive control of pET41/EGFP recombinant plasmid as well as a negative control, which contained no DNA. After being given our stock reagents, in order to form our own master mix we calculated the appropriate values for each reagent to make enough mix for a total of 20 ul for each PCR (71.4 ul nuclease-free H2O, 12 ul 1X PCR buffer, 9.6 ul 800 uM dNTPs, 6 ul 0.5 uM pAD1sense primer, 6 ul uM pAD1anti primer and 3 ul 2.5 units Taq polymerase). We then pipetted out 18 ul of master mix into each of our five tubes as well as 2 ul of each appropriate DNA template. We then incubated our samples in the thermocycler in the following parameters:
95°C for 10 mins
95°C for 1 min
56°C for 1 min
72°C for 1.5 min
4°C (hold, to suspend reaction/avoid degradation of DNA)
We then ran our 5 DNA samples through a 40 ml 0.9% agarose gel at 60 V before turning it up to 110 V to allow our DNAS to run faster.
Virual Cloning and Sequence Analysis
The purpose of this lab was to use online resources to determine the nucleotide sequence of our EGFP/pET41 recombinant plasmid, the primer locations and size of the PCR produce and to design a restriction digest experiment to future prove the identity of our expected DNA sequence.
To do this procedure, we used a variety of online tools. First, we used a nucleotide database to find the pEGFP-N1 plasmid nucleotide sequence. Next we used the WatCut restriction analysis tool to find the cut sites we used to cut the EGFP fragment out of the pEGFP-N1. To find the pET41(1) vector sequence, we used the LabLifeVector Database. Based on all the information and sequences we found from these databases, we then virtually “ligated” our EGFP fragment into the cloning site of the pET41(+) plasmid sequence to generate our entire recombinant plasmid sequence. From here, we searched for the location of our two primers, pAD1sense and pAD1anti, to determine the size of the PCR product.
To further confirm the identity of our product, we designed a restriction enzyme digest experiment using WatCut and two different enzymes that would cut. the PCR products appropriately and determined the sizes of the outcome of fragments.
III. Results
Ligating EGFP cDNA into pET41(a)+ plasmid
Figure 1. DNA ladder in lane 2, no results Lane 4-8
Lane 2 contained the DNA ladder and was the only DNA sample that showed up. Lanes 4-8 were also loaded with respect to Ligations #1-5 but do not appear in the picture.
Transformation of E. coli
Only two feint spots appeared on Dish 1. One green colony and one white colony (already circled and indicated for us) appeared in Dish 2 while five unspecified colonies plus 1 partial colony appeared on Dish 3. No apparent colonies were on Dish 4. Like Dish 1, there was one white colony on K2 but there were no green colonies. On K4, there was a large lawn of colonies as well as staggering speckles around the plate.
Miniprep of Plasmid DNA/Restriction Digest
Figure 2. DNA Ladder in Lane 1, nothing in lanes 2,3,4 and 7, possible bands in lanes 5,6,8 and 9
Although each lane in the gel was filled with its appropriate DNA sample, only the DNA ladder showed up prominently while the lighting of the photo makes it hard to tell whether there are lines in lanes 5,6,8 and 9. There appears to be nothing in lanes 2,3,4 and 7
Polymerase Chain Reaction (PCR)
Figure 3. DNA Ladder in Lane 2, nothing in Lane 4, one feint/one large band in Lane 5 and 6, two bands in Lane 7 and nothing in Lane 8
The DNA ladder was put into lane 2. Lane 4 contained the DNA sample from the colony that did not fluoresce green in the LB/Kan/IPTG medium and showed no results. Meanwhile, lanes 5 and 6 showed similar results (vague due to lighting of photo) of an obvious band and well as a possible feint band a little bit above it. The colony that fluoresced green found on the LB/Kan/IPTG plate was put into lane 5 and the white “unknown” colony found on the LB/Kan plate was placed into lane 6. Our positive control, which was isolated from a pET41/EGFP recombinant plasmid showed two obvious bands while the negative control in lane 8 showed no results.
Virual Cloning and Sequence Analysis
Based on the location of the pAD1sense and pAD1anti primer, the size of the PCR produce was 1183 nucleotides long, which matched the actual PCR product length. Using two new enzymes, Bpm 1 and NciI, we checked our results and found three possible digests.
If cut with just single BpmI digest, the three bands would be 910, 240 and 32 bp
If cut with just single NciI digest, the three bands would be sizes 496, 654, and 33bp
If cut with double BpmI and NciI digest, results would yield 5 bands at 496, 415, 239, 32 and 1bp.
IV. Discussion
Ligating EGFP cDNA into pET41(a)+ plasmid
Based on our lack of results, unfortunately none of our ligations were successful. This could have been due to the lack of glycerol we pipetted from the track dye since it might not have been properly mixed but it was most likely due to amateur pipetting errors since this was one of our first times using a micropipette. Through the process of forming each ligation or transferring the ligations into new tubes, we could have just been pipetting up nothing, which would mean that there was no DNA in the samples we loaded initially. The fact that the DNA ladder showed up means that the problem most likely did not have to do with how we formed our gel.
If our ligations had been successful, we should have seen two bands in both Lanes 4 and 5, one band in our positive control (Lane 6) and nothing in Lanes 7 and 8. In Lane 5, the first band would be higher up since we used a bigger ratio of EGFP than in Ligation #1 and the second bands would be around the same area. For our positive control, the singular band would be near the top. There should be two bands for Ligations 1-3 because our recombinant plasmids were cut by two restriction digests (NotI & NcoI), making two different sized pieces of DNA.
Since our positive control is uncut, it would be one large piece of DNA making it travel slower through the gel explaining why it would be close to the top. Because we know that the ligation was already successful done, this is our positive control because if nothing showed up, we would know that something could have gone wrong in other aspects of the experiment.
Since our first negative control had literally no DNA, there would be nothing to show in the gel. The ligation process was initially unsuccessful for our second negative control since no DNA ligase was added, explaining why nothing would show up in Lane 8 either. If something had shown up in these lanes, we would realize that some other factor was tainting all of our results.
If we had more time, hopefully our second trial would have been much more successful and we would be much more careful micropipetting each sample as well as making sure all the solutions we used were well vortexed.
Transformation of E. coli
In this experiment, we should seen results for plates 1,2,3, K2 and K4. Based on our results, most of our transformations showed semi-successful outcomes. Due to the possibility that some of our ligations were unsuccessful, white colonies (non-recombinant) and green colonies (recombinant) should appear on our 1:1 and 1:3 ligation plates. The lack of results on plate 1 could have been due to a mistake made in the formation of the ligate, since in a previous experiment using the same ligations, we yielded no results. Plate 3 contained our positive control ligation with uncut recombinant pET41a(+)/EGFP plasmid, and yielded a decent number of colonies. Plate 4, which had no DNA, showed nothing, which was expected. K2 was plated on a medium with no IPTG and yielded a single white colony (our unknown) while our K4 plate (LB plate) showed lawns of colonies. Since there was no kanamysin on this plate, plasmids that did not successfully ligate also grew since the kanamysin resistance gene played no part explaining why there were lawns of colonies on this plate.
Miniprep of Plasmid DNA/Restriction Digests
Based on the lack of results, it is feasible to conclude that there was a preparation error somewhere during the protocol, which could have caused the lack of DNA in the gel. Since the DNA ladder showed up, there is little possibility that the error had to do with the gel. We also might not have added enough track dye to our solutions, since there seems to be feint, but not obvious results of DNA in some of the columns. The lighting of the picture also could have been tinkered with if we had enough supplies to reprint the picture at different exposures. Regardless, our results were unfortunately inconclusive.
The expected results should have yielded one band for plasmids cut with a single digest and two bands for plasmids cut with two. The positioning of the bands would be based on whether the samples contained recombinant (green) or non-recombinant samples (white). The band for a single non-recombinant would travel a bit faster than a recombinant since EGFP is larger. The second band for a double non-recombinant would also be very feint and very small so there’s a possibility it wouldn’t show up clearly on the gel either. The undigested samples would run very slowly, since the DNA would be uncut and the size would be quite large.
If given enough time and supplies, a second trial would have yielded much more accurate and informative results. We would then be able to identify the results of our unknown (blue) sample of whether or not it is recombinant or non-recombinant.
Polymerase Chain Reaction (PCR)
Based on our results, our PCR reactions were all pretty successful. The outcome of the “blue” band also concludes how our unknown is most likely recombinant, since there is a distinct band very similar in size next to our “green” band, which contains a recombinant plasmid. Our “red” band yielded nothing, which is expected since it was non-recombinant, and our positive and negative controls yielded the expected results as well. A non-recombinant band wouldn’t show up very clearly since it would be very large and would travel very slowly down the gel. The fact that there were two bands in our “blue”, “green” and positive samples show that the ligation of EGFP was successful and that it was cut by two restriction enzymes.
Virual Cloning and Sequence Analysis
Through sequence analysis, it was concluded that the restriction enzymes BpmI and NciI could also be used as reagents for our PCR product. Based on the sized calculated, a single digest with either enzyme would produce two visible fragments (hypothetically) along with a third fragment of very little size that would probably not be seen on the gel.
Additionally, BpmI and NciI have restriction site with a nucleotide count of only 1 difference from each other. This would mean that a double digest with these two enzymes would not display the expected 5 fragments on a gel but only three since a single base pair would not show up on the gel and the 30 bp would probably be too small to show up on the gel as well.
V. References
Johnson, I.S., 1983. Human insulin from recombinant DNA technology. Science 219. 632-637
Prasher, D. C., V. K. Eckenrode, W. W. Ward, F. G. Prendergast, and M. J. Cormier. 1992. Primary structure of the Aequorea victoria green- fluorescent protein gene. 229-233.
Yang, T.-T., L. Cheng, and S. R. Kain. 1996. Optimized codon usage and chromosphere
mutations provide enhanced sensitivity with the green fluorescent protein. Nucleic Acids Res. 4592-4593.
by
shannihe (student)
