Hi, i'm having some trouble cloning a gene into a plasmid and I was wondering if anyone would be able to help?
I've managed to troubleshoot my problem and pinned it down to some incorrect PCR. I'll let you know what I did first of all:
- Double digest recipient plasmid, run on gel, purify, nanodrop.
- Design primers which are have ~10 bases 5' homologous for my plasmid and 3' 18 bases homologous for my gene of interest.
- PCR of my gene of interest using 300ng of plasmid template and two different (two different reactions that is, not 2 in 1!) Taq polymerases with proofreading. I used either 0.4 or 0.5 uM primer as recommended by each enzyme protocol. Cycles were according to each enzyme protocol.
- Gel purification of my PCR products
- Homologous recombination of my gene into the linear plasmid - two separate reactions, one for each polymerase.
- Transforming into competent E. coli recommended by HR kit.
- Pick 3 colonies from each plate growth, grow up in broth, make glycerol stock, miniprep the rest.
- Send some of miniprep product away for sequencing.
I've just got my sequencing back and it looks like my PCR has gone really strange. The first 14 bases of my gene are correct (14 out of 18 match with FOW primer), then I have 370 bases of nonsense that i've run an NCBI blast for and there is no known match, then my sequence resumes again from about 65 bases later and runs perfectly until the end of the gene. So 65 bases go missing and are replaced with 370 of random junk - even cutting out a section that the primer was binding to. My starting sequence and the point at which it resumed do have a small amount of homology (start = ATGGCCGCA, resume point = ATGCCTCC) but thats it.
Does anyone have any idea what went wrong with the PCR? It's strange that its happened with two separate reactions. One was an all-in-one mix and the other made up of separate components.
Thanks for reading