PCR cloning - (Aug/25/2008 )
I have cloned my target PCR product into pGEM-T easy vector and transformed into JM109 competent cells. After purifying the plasmid from several white colonies on the selective plates, I sent them for sequencing. However, interestingly, the sequences of my target PCR product were not identical but slightly different (some point mutation or indels but the length variation in within 8 bp). I am sure that the DNA amplified is a single copy gene on the chromosomes. Can anyone explain to me why? Any comment will be appreciated.
hello! During PCR the taq polymerase can introduce mutations, it doesn't happen if you use an high fidelity taq polymerase (I used Pfu DNA Polymerase from promega and it's perfect). If you want to know if this is the problem you can sequence your PCR products before cloning, but i'm almost sure that this is the problem.
Thank you. Lory. your answer may explain the point mutation but how about the indels? The length of inserted DNA from different clones are not exactly the same.
indels.... well DNA polymerase can slip. Thus the difficulty experienced when trying to PCR amplify triplet nucleotide repeat arrays and monoculeotide arrays.
If possible try avoiding them. Lower the extension temperature can sometimes help. Phusion can help a little, but not alot when the array is >500bp.
Can pfu DNA polymerase generate an overhang "A" base at the 3' ends of your PCR product ?
No, Pfu does not add an A. You can add an A to a Pfu product by adding Taq at the end of the reaction (assuming there is still some dATP present) or by setting up a Taq reaction with additonal dATP.