I am trying to synthesize CRISPR constructs, using Golden Gate to put my sgRNAs in the pHEE401E vector. Whenever I do colony PCR with my original primers, it appears that the reaction worked as I get a band of the correct size, however, when I sequence it I am not getting the right sequence! It appears to be showing me an empty vector. I am confused at how my colony PCR worked, showing my guide RNAs were there, but sequencing showed an empty vector.
Hello, may be you have phosphatase treated fragments which cannot ligate to anything else unless they meet another fragment that has a phosphate on the end.
Hello, may be you have phosphatase treated fragments which cannot ligate to anything else unless they meet another fragment that has a phosphate on the end.
Hello Malaika,
I agree with Bannikov's question.
Additionally, when you do colony PCR, you need to use one negative control and for this you can touch a tip on the plate without any colony (if already doing this, then skip my suggestion). This will give you high confidence whether a colony PCR result is false positive or not.
- Badges
- Science method