Insert being cut by E. coli? - (Mar/25/2007 )

Hi everyone,

I have a problem here in my cloning. I amplify my insert from cDNA with specific primers that I had designed with XhoI and XbaI cutting site which will be at the end of my PCR product. I digest the PCR product with the enzymes in Buffer D (both enzymes and buffer is from Promega) with no star activity stated. After I digest the product, I run gel and do gel extraction before I ligate the insert with my vector at 4C overnight.

After ligation, i transformed the construct into XL1-blue and TOP10 cells and incubate at 37C overnight. the next day i do PCR screening using the the same primer and the result showed the fragment is shorten from 1.8kb to 1kb. I try to extract plasmid and do RE digestion, and the result show one of the cutting site in my vector is gone. I later cut the empty vector with the same enzyme show the cutting is there in the vector......what is happening?

The TOP10 cells showed pin colonies even 24hrs incubation at 37C but control is ok. Does the insert is unstable or the protein is produced accidentally during the cells growth and it is toxic to the cells? how to overcome the problem?

pls help..............

johannes

I don't know what is happening with your PCR showing shorter bands. But the elimination of the vector cutting is likely due to the creation of a methylation site. For example, if your XbaI site, which recognizes TCTAGA as the cut site is surrounded by sequence ...gaTCTAGA... then you have created a GATC site which will be methylated in dam+ cell strains. Similarly for ...TCTAGAtc.. These won't cut, even though the site is still there, unless you clone in a dam- strain. A similar problem does not arise with XhoI.

You might also want to make sure that the PCR product is fully purified prior to cutting. Carryover PCR enzymes and dNTPs into the restriction digest can extend the 5' end of cut sequences and make the DNA unclonable, or in the worse case, clonable but with damaged cloning sites.