Hi, I transformed a PCR product containing GFP from pGFPuv without Lac promoter (change it with 50bp homologous arm for pRED/ET recombination) into Enterobacteria by electroporation. I expected to get GFP incorporated into the genome. However, PCR failed to confirm it. Then, when I checked the colony under UV light, it showed green fluorescent while negative control was not. My question is, is it possible for a linear DNA expressed? Or other possibilities of recombination into the genome in other positions? Thank you
In the case you described, no. It's probably either that:
1. You did get genomic incorporation and the PCR just failed for some reason (if it was colony PCR, it can be pretty hit or miss if it works properly)
2. The pGFPuv plasmid you used as a PCR template transformed into some of your cells and that's what it's expressing.
Maybe the DNA is present in your cells in plasmidic form and not integrated on the actual genome, or, for some reason it integrated elsewhere than you expected. You can perform a colony PCR extracting only a small portion of the GFP gene using proper restricion enzymes (thus knowing the exact size of the fragment) and check the product by electrophresis. If you get a positive output it means that the GFP is present in your cells; to know exactly where you then have to sequence (only if it is mandatory, if not sequencing, as you may already know is very time consuming)
Alexandra Johnson gave the most likely explanations. It is unlikely that you would get expression of linear DNA. One other possibility is that you have a mixed culture and some cells show a PCR product that predicts no integration.
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