Continuation of Three Way Fragment Assembly with Overlap PCR

 Introduction:

The previous week resulted in a gel that revealed 54 degrees annealing temperature for overlap PCR reaction 1, the reaction that is without primers responsible for binding the two homologous regions of left and tetr/a fragments, is the most efficient method for two way assembly product so far. Since 54 degrees Celsius was the highest temperature tested in the gradient experiment, it was decided to evaluate even higher temperatures to be able to fully visualize the full range of possibilities for optimal two way assembly with minimal ghost band products and high yield of desired product. The remaining amount of sample was loaded onto a gel for clearer visualization of last week's results, and then a higher heat gradient was conducted with left and tetr/a for the final time. Once a temperature is decided, the project can move forward by isolating the left and tetr/a overlap product so final three way assembly with the addition of the right fragment can be attempted in an effort to finalize a finished product before the end of the semester. 

Methods:

A 1.2% gel was ran with 1x TAE solution and 1 kb ladder with the remaining 15 microliters of sample from last week's heat gradient experiment (34 to 54 degrees Celsius). 

In order to run a new heat gradient from 52 to 70 degrees Celsius for left and tetr/a overlap, more left and tetr/a fragments were isolated from template DNA following Anna protocol. This calls for 10.75 ul of MasterMix, 0.5 ul of each primer, 0.5 ul of template DNA, and the rest of the volume up to 50 ul of PCR water. Left fragment utilized primers A and B, and used genomic DNA from Deinococcus radiodurans. Tetr/a fragment was isolated with primers C and D with E. coli plasmid as template. Both fragments were done in duplicates, and then 5 ul from each of the four tubes were ran at high milliamperage constants to confirm fragment identity, and then the remaining 45 ul microliters of each sample loaded individually into wells for gel excision and post gel cleanup with decreased volumes of dissolving buffer added to cut down on contamination of each sample. Each fragment cleanup was eluted in 30 ul of elution buffer and diluted appropriately for overlap reactions. 

Eight samples were conducted for the 52 to 70 degrees Celsius heat gradient with 50 ng of left fragment and 35 ng of tetr/a loaded into each, with 11 ul of MasterMix with a higher concentration of Q5 polymerase and a total volume of 24 ul. The overlap reaction with heat gradient settings was run for 15 cycles and stored overnight at -20 degrees Celsius prior to amplification. Amplification was done at 62 degrees Celsius, an increase of two degrees to account for the low yield seen in the previous heat gradient reactions prior to this week, in which Q5 MasterMix may be more effective two degrees higher than optimal for the primers as a general rule. The reaction was a total volume of 20 ul and ran for 30 cycles with a one minute long extension cycle at 72 degrees Celsius. 

A 1.2% gel 1x TAE solution was run with a 1 kb ladder for the heat gradient samples with 5 ul of each sample loaded. 

Results:

The first gel in figure 1 shows the repeated results from last week with more volume of sample loaded, which shows little to no change in visual results. 

Figure 2 shows the gel from the heat gradient of 52 to 70 degrees Celsius, which shows very little to no DNA in all of the lanes with the exception of some primer streaking towards the bottom of the gel. 

Figure 1: From left to right, Sample 1 (54 degrees Celsius), 2, 3, 4, 5, 6,7, 8 (34 degrees Celsius), "Mary" sample with left, tet, and right at 44 degrees Celsius. 

Figure 2: From left to right 1 kb ladder, Sample 1 (70 degrees Celsius), 2, 3, 4, 5, 6, 7, 8 (52 degrees Celisus)

Discussion:

The results from the heat gradient from 52 to 70 degrees Celsius was not ideal for obvious reasons, not being able to see the results and verify the results at roughly 54 degrees did not give us any clarity as to what temperature may be optimal. That being said, the results from the previous week at 54 degrees Celsius inspired enough confidence to move forward with that as the assumed optimal temperature, or minimally, one that works well enough for our purposes. It was deemed not important enough to verify method specifics as opposed to just attempting to get our desired final product. Given we decided to move forward, next week will be focused on creating more left and tet overlap product with a 54 degrees Celsius annealing temperature, isolating with gel excision and cleanup, and then attempting final three fragment assembly by introducing the right fragment. The right fragment will be attempted first at two temperatures, the snapgene calculation and at 54 degrees to mirror the left and tet overlap process. If needed, more heat gradients will be conducted for optimization similar to left and tet overlap.