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Position: Discussion forums > Bioscience biotechniques discussion forums > Molecular biology troubleshooting forum
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troubles with cloned insert
Posted by: agnes12 (IP Hidden, New member, 3)
Date: July 29, 2005 08:50AM
Hello, I would be very grateful for advice, here is my problem:
I have cloned 1,9 kb insert into pGEM-T Easy vector. After searching the clones I have sequenced cloned insert in few of them, but every time I find at least 4 substitutions. I have tested different bacterial strains: TG1 and DH5alpha and it makes no difference. Is it a question of mistakes generated during plasmid amplification in bacteria? Or is there anything I can do to prevent from these substitutions? (cloned insert isn't under UV at any of the levels of cloning)
Re: troubles with cloned insert
Posted by: hayes (IP Hidden, Unregistered user, )
Date: July 29, 2005 10:47AM
pGEM-T easy is for TA cloning of PCR product. The problem is because of limited fedility of your Taq DNA polymerase. Since 1.9kb is long and it is quite normal to have 0.1% misincopration during DNA amplification by the polymerase. I don't have the error rate of different DNA polymerase in hand but you can do a research on fedility of DNA polymerase for PCR. You should use high fedility DNA polymerase such as pfu, roches hi-fi long template kit etc to do a low cycle PCR. To my knowledge E.coli strain has nothing to do with the DNA mutation though it may affect methylation status. Best.
Thanks for Re: troubles with cloned insert
Posted by: agnes12 (IP Hidden, New member, 3)
Date: August 1, 2005 05:02AM
I was using TripleMaster Polymerase (Eppendorf), which is supposed to have 4-5x higher fidelity than Taq Polymerase. Now I'll try to use polymerase which has 50x higher fidelity.
Thank you very much for your advice!
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