Selective amplification problem in AFLP - (Jan/13/2006 )

Goodmorning,

This question concerns AFLPs, which are only recently performed in my lab, and so we are still perfecting our protocols. We are currenlty having the same, strange problem on two very different species. Curiously, we are having no problem getting to the pre-amplification step, with clear bands appearing predominantly in the 500-50 bp range that are perfect. However, selective amplification bizarrly produces useless smears predominantly in much higher bp ranges.

Somehow, we suspect that our taq is messing up somewhere, linking together chains or creating "blobs" of DNA, or at least that is what it "looks" like. We thought that this might be due to an oversaturation of the reaction solution with DNA, and reducing DNA amounts and the number of PCR cycles did really help the process for some samples, but we are still having very patchy results, despite apparently homogenous, and beautiful pre-amps. Has anybody out there had a problem like this before? Is there something obvious and stupid that I have missed? Any suggestions?

Sincere thanks to anybody who can help.... -Carey

I,
my name is Margot and I have the same problem

I make AFLP in the mosquito aedes rusticus and I use Eco and Mse.
On which species have you the problem?

The first time I have tried my protocol, I obtain very good and very reliable results.
After my pre-selective amplification I obtain fragments between 50 and 500 bp. But after my selective I have very big fragments and a little smear between 500 and 1000 bp. When I analyse my AFLP with the sequencer, only few bands with very low intensity appears on my profiles.
The more surprising is that in an experiment of 30/40 samples, there is often 1 or 2 samples what works. So I can think that my products are good (and I already changed all stocks).

I have tried several modifications of my protocol but with no better results:
- dilution 1/20, 1/100, 1/200 between my pre-selective and my selective
- I change the Taq : I usually use AmpliTaq gold and I try with AmpliTaq (Applied Biosystems).
- I test several primers
- I test a 1/20 dilution of my DNA before the digestion
…. And no results for the moment…..

I don’t understand why there is a problem in this selective PCR?
Don’t you think it could be a problem in the digestion or in the ligation that we can’t see?

If you have something new or some ideas, contact me

Good luck,
Margot