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| Molecular Cloning Forum DNA cloning forum. Discuss the cloning of plasmids, vectors, DNA, RNA, cDNA, proteins, and other molecules for expression, sub-cloning, and other methods. Also discuss digestion, ligation, transformation, plasmid preparation, cDNA library screening, and competent cells here. |
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#1
11-17-2009, 10:34 AM
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 primer-ligation relation Is it possible that if I have not included 6-8 bases near the RE site during primer designing, I can have trouble during ligation? if the RE is not able to cut properly , then my insert and vector wont ligate well? I am having trouble with my ligation. I use 1:3 molar vector:insert ratio, 50 ng of vector in the system. I also dephosphorylate the plasmid.. I have tried overnight ligation at 18degree C and rapid ligation at 22 degree C for an hour if i transform only the plasmid i get colonies.. i also purify the pcr products so my cells are OK the only problem i see is that the ligation is not happening is it faullty primer design?? am i stressing out too much and is it something simpler which is causing the problem ??  any suggestions??  |
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| primerligation , relation |
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