digestion problem - incomplete digestion of transformed plasmid (Jun/30/2005 )

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hello,

i am in the process of screening my putative clones. i had done pcr colony, and some appear to be positive. i try to confirm the clones using extraction and digestion with the same restriction enzyme i used to ligate both insert and vector.

the first time i digest, none of the transformed plasmid were digested (the band appeared to be exactly the same size as the undigested). assuming the plasmid were too concentrated, or the enzyme used was not enough to cut, i reduce the volume of undigest (less concentration), and i increase the volume of enzyme used(which was 0.5ul to 1.0ul), but nevertheless, the result still the same-the band appeared were the same size like the undigested plasmid.

is there any possibility that the site (i used SalI to cut, both vector and insert), was mutated?

help me, i'm desperately need to get the right clone!

Hi keyrana,


The fact that it does not digest at all (ie. runs same as undigested vector) suggests that something has happened to the site. I have a theory with no evidence other than seeing this problem before that ligating results in a structural change to the site which inhibits recognition by SOME enzymes (similar to methylation).

Is there anyway of sequencing one of your positive clones, that way you can be sure if the insert is there and if the restriction site is intact. Or digesting with something else that cuts within your vector and insert to be sure that the PCR didn't give a false positive.

Cheers,

Scott