I'm having trouble with contamination of linearized plasmid with the intact circular plasmid. I have heavily overdigested the plasmid, gel purified, and digested with another restriction enzyme, but I still get PCR products over the cut site. This is a problem because I'm trying to do PCR using a bridging oligo which introduces a necessary additional sequence into the cut site, and it seems that there must be no intact plasmid present for this to work.
The problem you are facing is connected with the low effectiveness of the digestion. Therefore the cut site is still present after purification step.
Cheers!
Dear Andrei, RF cloning seems to be a very interesting technique and I will remember it for future projects, but I'm afraid it doesn't solve my problem in this case. The cloning procedure would be simple enough using restriction, but I want to skip cloning altogether using only PCR. I guess I could in principle generate the PCR template using the RF protocol, but probably the same contamination problem would occur there? I mean, DpnI digestion is probably not any more perfect and total than my current digestion protocol.
Dear Maciej, that is exactly the problem I'm trying to solve. The digestion is so perfect that I can see absolute no trace of the intact plasmid on gel. To be sure I gel purify it anyway. But despite all this there's still enough intact plasmid left to function as a PCR template.
Hello again Andrei! I got excited about the very clever idea of methylation-sensitive digestion, and thought I could apply this to "fix" my PCR contamination problem. But it turns out that DpnI needs a restriction site which is unfortunately not present in the essential region of my plasmid, so I should still go to cloning, which I'm trying to avoid in the first place. Do you know if there are any other restriction enzymes which cut only methylated DNA but have a different recognition sequence?
Please take a look here http://epigenie.com/guide-key-restriction-enzymes-for-dna-methylation-analysis/
Maybe you will find here some additional enzymes.
Another thing that comes to my mind is the HPLC. Have you considered this step after digestion?
- Badges
- Science topic