Low PCR product for antibody gene - (Mar/11/2006 )
Dear all,
I amplified heavy chain by using PCR and I got very less product. I tried to increase the product by increase template, primers, Taq , dNTP and did gradient of annealing temperature, but I got the same result. Anybody can give me any suggestion ?
Thank you,
you dind't try longer elongation times...
and did you try more kits?
What kind of primers are you using to amplify your heavy chain? You should use primers that anneal to the type of heavy chain you're dealing with i.e. IgG1, IgM, etc.
and did you try more kits?
you might also want to try increasing the number of cycles.
Thanks for all suggestions.
I am working on phage displayed scFv library construction. I got cDNA from human B-lymphocytes of 70 non-immunized donors and I amplfied heavy genes and light gene using specific primer sets for all human antibody families. I got sufficient amounts of light chain products whereas heavy chain were not reached the same yield. Both heavy and light chain showed non specific band which was smaller size than the predicted size and was higher amounts in heavy chain.
I already tried longer elongation and touchdown PCR , non specific band tended to decrease but I got the same less amounts of products of predicted band. I used the Expand High Fidelity PCR system and I got less products compare with Taq polymerase. I don' t have an experince in RT-PCR. I have no idea what should I try next ? Is it possible that cDNA was not good quality ? but I got high yeild and quality of light chain. I used MMuLV, superscripII, iscript reverse transcriptase for amplifying cDNA.
I hope I will get any suggestion
Thank you,
Potjamas