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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
11-17-2010, 04:45 PM
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 Inverted insert Hello everyone I tried to clone a 750 bp insert in a 5 kb vector, I did it the old fashioned way as following: Cut the vector and insert using ECON1 and Nru1. Dephosphorylation of the vector using the CIP, transformation, I got many cultures. after that I amplified the miniprep using insert specific primers and all looked good on the gel. after DNA sequencing I found that the vector has my insert but it was cloned inverted into the plasmid !!!!! Does anyone have an idea why did this happen??? thanks  |
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| insert , inverted |
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