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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.


Large Insert, successful ligation, no clones with insert

Large Insert, successful ligation, no clones with insert - Molecular Cloning Forum

Large Insert, successful ligation, no clones with 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 04-28-2011, 08:09 PM
Pipette Filler
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Unhappy Large Insert, successful ligation, no clones with insert



I am attempting to clone a 4800bp insert into a 4900bp vector. When I run the ligation reaction on a gel next to a control with both insert and vector with no ligase, it is clear that there is little to no insert or vector alone left in the ligation, and bands appear where there should be a ligation product (the control shows only the bright band of insert and vector). So it appears that the ligation was successful. I then transform the ligated DNA into XLIB and get colonies, but after countless minipreps none seem to have the insert. Using CIP on the vector before hand produces absolutely no colonies at all, leading me to believe that it may have degraded the DNA in some way. I'm currently trying again with Antarctic Phosphatase. Any suggestions? The vector I'm using is pRS416, as I need a ura marker.
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