Digestion problems with BamH1 - .... in a linearised vector.... (Jul/16/2008 )

Hello all,

I've a problem. I'm trying to clone a new lentivirus vector. Fot this I've a good working vector with eGFP. Now, I try to remove the eGFP and clone in a "triple-fusion"-Gene (RFP - HL - TTK). But I've problems with removing either the promotor or the eGFP. I need to cut the vector with EcoR1 + BamH1 or Sma1 + BamH1. Each RE is working very well alone (always linearised vector). But if I do a double-digest or a step-by-step digest (with checking the result of the first RE on a gel) the second RE is not working any more. There is no DNA-fragment which is falling out.... (should be arround 600 or 750 bp).

I've no idea why the second RE is not working (doesn't depend on which enzyme I use).
I tried different buffers, different amount of enzymes, DNA, BSA, Buffer, water, etc

Do you have any idea???

Cheers,
Kathy

Hi Kathy,
I know this problem. After we changed our supplier to the cheaper restriction enzymes we also had problems with double digests or trying to clone our digests... suddenly it worked much poorer. It took us a while to find out that it were really the enzymes - believe it or not.
So we changed back to the RE from Roche Applied Science. They are a little bit more expensive (depending on the enzyme) but they have the strictest quality control and the enzymes are much more purified than those from the other suppliers and you usually need less enzyme and reactions until it works... so in the end more cost effective...
For your double digest with Roche RE use EcoR1 + BamH1 in SURECUT Buffer B.
If it doesn't work with the Roche enzymes there must be a mistake within your experiment.
Wish you much success...

Hi Senior_Scientist,

we are usind RE from NEB. I thought, they are good. Maybe we should try them from Roche, perhaps they can send us a small sample, just to try it once.
In fact, that all Enzymes are working within a single digestion I guess, that the experiment is ok.

Something new: A double digest with enzymes cutting 5000bp away is working. Only the double digests are not working, which have to cut within a defined region (1000bp).

Thanks, for answers.

Cheers Kathy

My guess is that your sequence is not what you think it is, and these enzyme cut sites are next to one another. I'd suggest that if you continue to have problems, that you sequence the construct.