PCR Colony Tests

PCR colony tests are a technique I used to help determine if the construct we created via restriction digest or CRISPR-cas9 genetic engineering were successful in becoming sequenced with the correct fragments.

A PCR colony test begins with a plate of cultured bacteria so that distinct and "juicy" colonies are abundant. The fact that the colonies grew at all means that the DNA was successfully incorporated into their genomes via bacterial transformation, because they grew despite the antibiotic meaning they contain resistance. The question is whether the DNA we constructed is in the correct sequence to be used in the valuable NSCs. First, colonies are swabbed one at a time with a p2 pipette tip and pipetted up and down in ultra pure H2O. Then, specific volumes of these mixtures (about 20 colonies are commonly tested at a time) are pipetted into a stock of reagents necessary for PCR. The PCR temperature settings for a PCR colony test are slightly different than for normal PCRs because they must begin warmer so that the bacteria are completely lysed open and the DNA contents free. This was partially achieved through pipetting up and down. The amplified DNA is then run through gel electrophoresis so that the fragment sizes can be observed. If none contain the desired bp length (which is unfortunately, often the case), then the DNA fragments are re-engineered, maybe in the same way, or maybe using a different method. However if a colony contains the correct number of base pairs, this DNA can be amplified further and sent for sequencing. If the sequencing comes back with the correct sequence, the DNA can be re-transformed, then grown up for a maxi or mini prep so that it can be used in a viral prep to transfect the NSCs to be used in vitro, or in vivo analysis.