Digesting PCR products and cleaning up digestions - a few quick questions (Oct/26/2005 )
Is there any way to verify digestion of PCR products (amplified with primers that introduce the appropriate restriction sites and the extra bases upstream of those sites for digestion)? Basically you're only digesting 6 bases or so off of the end of your insert. I'm guessing that one just digests for an hour and assumes that everything worked out. Since my insert is 532bp I won't see a 6bp difference on a gel.
I also have a question regarding my vector. Basically I'm cutting out a 76bp band from the MCS. I've been gel purifying the digested band of interest, but I'm getting horribly low recoveries from the particular kit that I'm using (I think we got a bad batch of columns or something because everyone else in the lab is having this problem). If I phenol:chloroform/ethanol precipitate the digestion will I get rid of that 76bp band, or will it stick around and eventually compete with my insert? I suppose I could run the digestion through some sort of sephadex column as well. Any suggestions?
As far as whether my PCR products are actually digested, I just take it on faith (plus the fact that I usually digest my vector at the same time and run it on the same gel, so I have an idea my enzyme was good)...
EtOH precipitation will not remove that 76-bp piece, and if you can't get it out of your reaction, it'll certainly cause problems. A few suggestions:
1. Borrow some good columns from another lab.
2. Use a non-column based method of recovering your DNA from the gel (freeze-squeeze, electroelution, ß-agarase digestion, glass milk, etc.).
3. If the small fragment is coming from a MCS, maybe you can cleave it with another enzyme that doesn't appear elsewhere in your vector? Not sure this would be of tremendous help, though...
Thanks for the quick reply!
Yeah, I was pretty sure about the PCR digestion thing.
Actually, 5 minutes after I posted this topic I was poking around and stumbled upon an unopened Promega Wizard SV PCR clean-up/Gel-purification kit (the 250 prep package)!!! I was previously using a Qiagen kit.
I swear, there are so many overlooked and wasted things in my lab. I am certain nobody else knew that we even had this kit. Just like the boxes and boxes of outdated DNA mini/midi/maxiprep kits.
Anyways, I've never heard of electroelution, so I'll look that up. How efficient is the freeze-squeeze?
It used to work pretty well, before column based kits were around. There was another way, too, that involved putting the gel slice in a microfuge tube with a pin hole in the bottom, and placing this in another microfuge tube, and spinning the whole thing to collect the liquid. I haven't used any of these procedures in years, but they used to work okay way back when...
BTW, I was coming back on 'cause I thought of something else -- the Qiagen kit is very pH dependant; maybe someone made your TAE or TBE incorrectly? If the pH of the melted gel slice + guanidinium thiocyanate solution (buffer QB) is >7.5, efficiency of binding to the silica membrane drops off dramatically. You can adjust the pH with a little 3M sodium acetate, pH 5.0 if needed.
Yeah, I read about the pH dependency of the Qiagen kit. I checked out the buffer beforehand and the pH was right (5.2'ish), but I didn't consider the gel slice affecting the pH. That QB buffer has a pH color indicator though, and it looked fine. Maybe I'll just check the pH later when I go to troubleshoot that kit.