Spurios errors while cloning - cloning mutations (May/22/2006 )
I am trying to PCR amplify out a region of two proteins currently in a mammalian expression vector. each and put it into a pGEX vector. These two proteins are highly homologous, even at a DNA level. When I do this for one of the proteins (say protein 1), it works just fine. I am able to amplify it (using Pfu ultra), cut it with EcoR1 and put it into the pGEX vector. After sequencing, the correct sequence is there. When working with the other plasmid (say protein 2), I amplify it (again with Pfu Ultra), cut with BamHI and EcoR1 and ligate. When sequencing this protein 2, however, there are lots of spurious errors in the sequence. In fact, every clone I have made/sequenced has introduced an unwanted error.
One thougth, is DMSO a culprit? For some reason, protein 1 "doesn't care" if DMSO, but the problem protein 2 requires DMSO in the reaction to PCR amplify. Any suggestions for better extension?
Leigh
Are the errors similar in any way?
Is it possible is contained within GEX?
Try using Betaine instead of DMSO (my recommendation).
-Matt
If your construct is toxic, you may be selecting only incorrect mutant clones during growth.
Is it possible is contained within GEX?
Try using Betaine instead of DMSO (my recommendation).
-Matt
There are two sets of constructs using the same backbone amino acids, but one set has an intentional point mutation. In the wild type version, the error (both times) is at the N-terminus. In the 'intentional point mutation' version, there are errors at the C-terminus (both times). The spontaneous errors are similar within each background, but not identical.
No, it does not seem to be an issue with GEX. I will try using Betaine.
Thx,
Leigh
Not that I am aware of. It does regulate a phosphatase, but I'd be surprised if that was an issue for bacteria-- particularly DH5alpha, where it shouldn't produce much protein.