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AFLP
Posted by: Sabine1 (IP Hidden, New member, 1)
Date: July 19, 2005 08:42AM
I’m working with AFLPs and I had already quite good results at the beginning of my work but after about twenty samples (with different primers) there came out only a few peaks at the beginning! I suppose there could be a problem with the restriction-ligation because I’ve tried to control the different steps of the AFLPs, doing parallel preparations with samples that have already worked and therefore I think the problem has to be in the first step! I’ve changed all my reagents with new ones but there was no improvement! Can anyone give me some good advice or did anybody have the same problems?
Re: AFLP (I also need help!)
Posted by: mamitt (IP Hidden, New member, 2)
Date: March 3, 2006 07:43AM
Hello, Ms Sabine1 (and others people!):
I just found your post while I was serching for any help with my AFLP problems. Sorry, I cannot help you out, but I was wondering if you solved your problems as my broblems sound quite similar to yours. I had good results for the first 100 samples with 3 different primer combinations, but suddenly, I started having very low intensity bands (or even no bands sometimes). I am using ABI AFLP kit, and I changed all kits with new ones, but I still cannot see any improvement even with the DNA which once had successful results. I suspect something is wrong with my restriction-ligation. My college, who had the same problem (sudden failer of AFLP), recently changed from low concentrated restriction enzyme to high concentrated one (EcoR1), and it worked. Presumably, larger amount of glycerole contained in low concentrated enzyme prevented the restriction-ligation to work. (However, this didn't make any difference for me.) So, you can think about this if you use the low concentrated enzyme. Anyway, if anyone could give me some suggestions or advice, I would be very appreciate it!
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