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ligation problem
Posted by: porn (IP Hidden, New member, 1)
Date: October 31, 2007 01:36AM
I try to subclone 1.5 kb fragment to pET vector. Both plasmid (plasmid containing fragment and new vector were cut with NdeI and BamHI. I use double digestion and then purify form agarose gel. Then ligate using Ligation high (TOYOBO, Japan), but no bacterial colony after transformation. I try many vector:insert ration but still not sucess. From the prvious posted topic that has similar problem with me, someone suggest to use PCR purification method replace agarose gel purification. I wonder that can PCR purification method separate fragment that I want to insert in new vector form previous vector. Thank you in advance If someone can help me or suggest the other way to solve my problem.
Re: ligation problem
Posted by: sammer (IP Hidden, New member, 1)
Date: November 14, 2007 02:20AM
i'm trying to clone 2kb fragment in pET33a vector. both plasmid and vector were double digested and purified using agarose gel purificatipon system. ligate using T4 DNA ligase and then transformed. no colonies observed and once i got results but they were consider as nagative because the colony has the vector that was just like as original or uncut vector.
Re: ligation problem
Posted by: HCscientist (IP Hidden, New member, 3)
Date: February 1, 2008 06:35AM
You can try to ligation-independent cloning strategy. It is quick and high efficiency.
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