Purpose: To isolate the colonies in which LuxOD47A on psB1AC3 was successfully transformed
Protocol:
We conducted a colony PCR of the colonies that grew on the overnight plates as a preliminary verification that our LuxOD47A BBk sequences on pSB1AC3 plasmids were properly transformed into the cells. We prepared tubes for the six largest colonies from each sample (initially formed with EcoRI and XbaI), for a total of twelve colony PCR tubes. As this process is only for verification (as opposed to LuxOD47A BBk formation from the gradient PCR with gene-specific BBk primers), the high fidelity afforded by the Platinum Pfx polymerase is not necessary, so the less-expensive and sufficiently-reliable Platinum Taq polymerase - and the corresponding Taq PCR buffer, with MgCl2 instead of MgSO4, was used instead in the Master Mix. Forward and reverse LuxOD47A-specific primers (without BioBrick sites) were used in the sequence amplification. We completed the Master Mix by adding dNTPs and double-distilled water, and then evenly distributed 650 uL of it across thirteen tubes (twelve for the colony testing and one negative control), each containing 50 uL aliquots of Master Mix. To add DNA to our relevant tubes, we delicately touched a pipette tip to a colony of interest and immediately inserted the bacteria-infested tip into the respective PCR tube. This was done for every tube except the negative control, which only contained Master Mix.
We then proceeded to run the colony PCR. Unlike the gradient PCR, the annealing temperature does not vary between wells and is set at 5 degrees C below the primer annealing temperature of 60 degrees C. The purpose of each step in the process is consistent, however. The Platinum Taq polymerase was activated in a 6 minute initialisation step at 94 degrees C. 36 cycles of a denaturation step (30 seconds at 94 degrees C), an annealing step (45 seconds at 55 degrees C) and an extension step (90 seconds at 72 degrees C). This was followed by a 10 minute final extension at 72 degrees C, and held at 4 degrees C once the PCR was complete.
Results: These are shown below. The bands are an appropriate size, but the negative control lane is not distinct from the other lanes. We progressed anyway with our restriction digest verification. Restreaks were also made for each of the colonies that were tested in the colony PCR.
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