DGGE - PCR and gel conditions - (Jul/01/2007 )
for the past two weeks i've been trying to setup and run our DGGE properly (and as no one in my vacinity has ever used it, I have to learn all on my own).
My DNA is extracted from coral mucus and I amplify using 341F-GC and 907RM (touchdown PCR, annealing 65-55 Celcius). running the product on an agarose gel gives a nice product of ca. 500-600bp (which is expected, I do see primer dimers but I think i should't worry about those as it should run right through my gel). so far so good. however, when I run my products on a 6% gel, using a gradient of 20%-65% (urea-formamide) all i'm getting is a smear (around 35-55% in the gel). I can't seem to be getting proper bands... i've loaded varying amounts of DNA.
As I'm running other samples (that were amplified by someone else... using the same primers, but not the same stock) we are getting nice bands (not the best, but still - bands). That's why I'm starting to think my problem is with the DNA prep. or PCR.
So, my questions are as follows:
1. should I prepare the DNA with nested PCR - first amplify the whole 16S gene and then just the part I need for DGGE with the gc-clamp? what are the pcr conditions you are using?
2. would it be better to extract the DNA (after PCR) from the agarose gel and use that for the DGGE run?
3. anything else i'm doing wrong? amount of DNA to load on the gel etc.
I'd appriciate any pointers or what not you can give me.. I'm a little lost here.
I use touchdown PCR 65 - 49
30% - 60% gradient
I amplify a 400bp fragment using gc clamped primers
10 microlitres of the pcr product is run on the gel overnight at 80V
This gives us nice bands
To get nice bands in a DGGE you can:
1) Make shure you only get ONE defined band in your PCR, try increasing Mg++
2) After PCR I extract the PCR with phenol, not necesary to purify from gel.
3) When pp I do not use NaAc, I rather let pp overnight.
4) For DGGE I use 8%, or you can even use 10% for nicer bands.
5) There are other things you can change to your DGGE to have a better resolution, try diffent gradients, increase running temperature, time, DNA concentration.
6) BE PATIENT
For any other detail feel free to e-mail me; I'll be glad to help.