Continuation of Three Fragment Assembly Using Overlap PCR

 Introduction:

The previous weeks have led to confusion surrounding the ideal temperatures for annealing PCR conditions The most recent gel which included a 34 to 54 degrees Celsius heat gradient to evaluate the optimal temperature for left and tet fragment assembly resulted in a gel with no DNA visible and no primers. There was suspicion surrounding a mixup of tubes during that process which would explain the lack of DNA, since that result had not happened before amongst all of the trials this semester. A preliminary test was done amplifying another set of tubes which could have been mixed up with the tubes actually amplified the previous week, to check for any results. Afterwards, this week we carried out a re-do of the previous week's experiment and got partial clarity for steps moving forward. 

Methods:

Due to the suspicion of two sets of eight tubes being confused with the other set of eight, which were amplified and ran to reveal no gel results, the tubes labeled "Overlap reaction1" were amplified at 60 degrees Celsius and ran on a 1.2% gel with 5 ul of sample in each well. This gel showed no signs of DNA so the entire experiment was decided to be redone. 

The left and tet fragments were isolated from genomic D. radiodurans DNA and E. coli plasmid samples. Each fragment was done in 50 ul reactions, with primers 1 and 2 and 3 and 4 respectively at 60 degrees Celsius annealing for 30 cycles. Each fragment was loaded in its entirety onto a gel and excised for cleanup for overlap steps. 

Overlap was done loading 50 ng total of the left fragment and 35 ng of tet to achieve approximate equimolarity according to the Nanodrop One. Eight samples were done for the heat gradient of 34 to 54 degrees Celsius for 15 cycles, each with 11 ul of MasterMix, 5 ul of left, 7 ul of tet, and 1 ul of PCR water. They were flicked and pop spun to ensure homogeneity and extensively labeled to avoid possible confusion of samples. 

An additional sample, the "Hail Mary" (aka Mary) included 7 ul of left, 5 ul of tet and 7 ul of right fragment to again achieve an equimolar ratio of the three (50 ng left, 35 ng tet, and 50 ng right). Mary was ran at 44 degrees Celsius for 15 cycles for overlap based upon the results of the gel seen two weeks ago, in which 44 degrees produced the strongest band of product with the least amount of ghost banding. 

The eight heat gradient samples and Mary were amplified at 60 degrees Celsius for 30 cycles with 1 ul of each primer, primers 1 and 4 for left and tet overlap and primers 1 and 6 for Mary. The amplified samples included 5 ul of overlap reaction 1, 1 ul of each primer, 10.75 ul of MasterMix, and the rest PCR water up to 20 ul. 

All samples were ran on a 1.2% gel with 5 ul of sample loaded into each well in a 1x TAE solution and a 1 kb ladder. 

Results:

The first gel to test the mixing up of tubes from the previous week resulted in a gel which also showed no DNA and no evidence of primers. 

Figure 1 shows the results of the gel from this week, confirming overlap products of approximately 1700 bp in each of the eight lanes, some more faint than others. The Mary sample left a large smear and no apparent bands. Primers are seen in only some of the lanes. The amount of DNA visualized is still very little in comparison to previous gels. The two temperatures that had the best overlap product bands were 34 degrees Celsius, and 54 degrees Celsius. 

  

Figure 1 (from left to right) 1 kb ladder, 54, 52.3, 49.7, 46.3, 41.7, 37.7, 35.3, 34, and Mary. The strongest overlap product band was seen in lane 54. 

Discussion:

Although not a large amount of results, it is seen from the the redoing of this experiment that definite overlap occurs at a wide range of temperatures, but arguably the best at 54 degrees Celsius. At 34 degrees Celsius, which has the second best band, there was clearly overlap but less overlap product as compared to 54. It is believed that 54 degrees may be optimal in making product and decreasing prevalence of any ghost bands, but it is very difficult to definitively visualize due to the lack of DNA in nearly all the lanes. These samples will be more accurately seen by loading the remaining 15 ul of sample on a gel to attempt seeing ghost bands and stronger bands from each lane. The Mary sample is a long streak that shows no specific bands, which may be attempted at 54 degrees Celsius the next week. With 54 degrees Celsius apparently being the best this week, that leaves two major questions. For one, another heat gradient will need to be done, potentially from around 52 degrees to about 68, just to finish the full range of temperatures considered plausible. The next, it is concerning that Snapgene had once told us that 36 degrees Celsius was supposed to be optimal, which seems to be clearly not the case. It indicates that large heat gradients may need to be done in the future with other projects as well, just to ensure optimization. The other major concern from this gel would be the lack of DNA product post PCR amplification, which has always been more than sufficient in the past. It seems that the current PCR conditions are suddenly not as efficient for amplification, which may be due to issues with the MasterMix, but that is not confirmed. If low DNA output from PCR is still being seen this week, it may be investigated to be sure that there are no issues with any part of the process that would inhibit product amplification. Thankfully though, redoing this experiment from last week did not result in another empty gel, and some results, although faint, could be seen and utilized for some hopefully successful next steps.