Hi Everyone,
I am working on ATG-protein nucleotide sequence in pCMV6 plasmid construct. In this construct I inserted desired sequence to extend the loop using by NEB site-directed mutagenesis kit. The desired sequences were inserted, but an extra nucleotide (12 nucleotide sequence) was found before the ATG sequence start region. How to troubleshoot this issue?
First I would check your primer sequences and make sure their are no alternative primer binding sites. Is the sequence insertion toxic to cells?
Have a look at the additional 12 nucleotides sequence; is it coming from your oligos? maybe your oligo have a sort of repeated sequence.
you can increase the hybridization temperature of your PCR (up to the primers tm and even higher if you are desperate), reduce the number of PCR cycles (12 to 15 is enough for site directed mutagenesis) analyse several clones.
- Badges
- Science topic