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No Plasmid digestion - Enzymes don't cut my miniprep Plasmid-DNA (Aug/02/2005 )

Hi,

I have a problem with my cloning. I have done every step of the route, from preparing the vectors by cutting with appropriate enzyme, cleaning, SAPing. I have cloned the insert first in the pGEM-T-easy vector (TA-cloning with PCR-product) and cut it out from there. I cleaned the insert and ligated it with the destination vector and transformed it into different E.coli cells (HB101, JM109, C600, Topo10) and get colonies which looked well.

Now, the problem is: I'm doing my normal alkaline lysis minipr├Ąp (which always worked well), get DNA, but the DNA will not be cut by different enzymes (BamHI and SmaI as singel digestions). I have tried the RE on Plasmids, the worked all well, just not on my miniprep DNA. I get three bands, the same pattern for my digested DNA as well as for a non-digested control. Looks like unidgested plasmid DNA (coiled, supercoiled, relaxed). Normally, the vector should be minimally be linearized, but nothing to see.

Anybody got an idea what's going on? I'm really frustrated! sad.gif

-biomaus-

If you add plasmid DNA that digests well with BamHI and SmaI to your miniprep plasmid do you still get digestion? If yes then you know that you don't have a inhibitor problem with your minipreps, if not then you do.

Daniel

DNA sequencing software

-Daniel Tillett-

Thanks, Daniel, I will try this immediately!

-biomaus-