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[TDC_Tiger]

Hey,

This is my first post on here, and I hope you all can help.

I've been attempting to insert the 7.8 Kb Cyp1B1 gene into a 5.2 Kb plasmid (called pCMV-3tag-6, which can be found on page 8 of this site: http://www.genomics....nual/240195.pdf). I have been digesting the plasmid and pcr product of Cyp1B1 with Hind3 and EcoRV, and after each digest, I denature the enzymes at 80C for 20 min. I know that the digest for the plasmid is working due to controls that I have done (ligation efficiency after single cut digests and double digests). However, I don't have a test for a digest of the pcr product. For some reason though, I cannot get the insert into the plasmid. I know that the ligase works and I've tried a number of different insert to vector molar ratios (from 3:1 --> 10:1), but nothing seems to work. I have even tried inserting much smaller pieces (300bp which have been digested from much larger pcr product). For some reason I am having extreme difficulty ligating anything into this plasmid. Any suggestions?

Thanks,

T.D.C.