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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.


Inverted insert

Inverted insert - Molecular Cloning Forum

Inverted insert - 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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Old 11-17-2010, 05:45 PM
Pipette Filler
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Angry 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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