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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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01-09-2008, 01:41 PM
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 DNA Ligation Problem Double Digestion Hi, I have a new problem I didnt get colonies after transformation.  I cloned a 1.5kb DNA piece into the vector/plasmid MCS pcDNA3.1+ however, but when I tried to put another 1.5kb piece into the MCS I cant get any colonies. I am conducting PCR on the piece, then I double digest the PCR product and the plasmid by double digestion with BamHI and HindIII overnite at 16C. I then conduct ligation at room temperature with the T4 DNA ligase overnight. I am ligating at a ratio of DNA to Plasmid at about 3:1. And the total amount of insert use for transformation is 5ul/50ng DNA. What could I be doing wrong? thanks        |
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