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white-green selection problems


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#1 mark.ibt

mark.ibt

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Posted 22 January 2015 - 09:30 AM

Hi guys,

I have a problem with white-green selection system.Please, give me some advice.

I have one gene with the length of 2.0 kb and I wanna clone it into PCR2.1 vector (Topo) to sequence it. I performed ligation reaction follows manufacturer's protocol (14 degrees, 4 hours) then I transformed ligated product into Ecoli DH5 anpha and plated on solid LB media with amp and Xgal. The result was that, I obtain the whole white colonies in petri LB media and the number of colonies is enormous. I carried out direct PCR with specific primer for the gene but no band was seen. I extracted plasmid and cut with EcoRI enzyme, i got surprised result- DNA plasmid wasnot cut by EcoRI. Please give me some suggestions, how I can do to clone successful the gene and help me explaining the result above. thanks a lot.






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