Transforming after digesting PCR product - (Sep/14/2006 )
I ran a PCR reaction the other day for a single-point mutation (QuikChange protocol). After digesting them with Dpn I, I put the reaction tubes in the freezer overnight (I had to leave). The next day, I ran an agarose gel (attached) to check whether the reactions had worked and everything was at the bottom (they should be 6 kb, which is more-or-less at the top).
My question is this: does the fact that I left my reactions in the freezer overnight explain my shoddy product? If so, how could I store my PCR product before I transform it? If not, does this look like a familiar problem to anyone?
Thanks in advance for your help. This forum has been quite the valuable tool for me.
Hi there!
To me it looks if your PCR has not worked at all...the things at the bottom look like primer dimers..
Stardust
To me it looks if your PCR has not worked at all...the things at the bottom look like primer dimers..
Stardust
I was thinking that, too. Thanks.
It can be very difficult to see a quickchange product (see quickchange protocol from stratagene). What you see at the bottom looks like primers. We always precipitate the DpnI digestion, resuspend in 10ul of H2O and then electroporate 0,5ul. I would try to electroporate anyway. At least you can be confident you don't have parental DNA in your digestion...
you might not be able to see your PCR product after 12 or even 18 cycles. So, transform your cells anyway
The attached picture shows a (sloppy) gel that I ran a few months ago using the same protocol and plasmid. You can see a band in the 6 kb range in two of the reactions here. With this in mind, would you guys still recommend that I transform?
Depends on how valuable is your competant cells and your time. AKA How desperate are you?
Usually I am pretty desperate. Concentrate down your sample and transform. And pray that the gods of science (?), umm.... the goddess of probability and uncertainty is looking favourably on you.
But also consider that your reaction has failed. If your gel was supposed to look like that, it doesn't look good. Go back to the drawing board and consider what might have gone wrong or gone off.