problem with faint bands

chovek69 ivanoov at gmail.com
Wed Aug 30 08:03:34 EST 2006


I have quite a lot of experience with AFLP and I can say that the
thermocycler program is a critical parameter. At first I had similar
problems like you but then I optimized the cycler regime acording to a
manual by Applied Biosystems
http://docs.appliedbiosystems.com/pebiodocs/00402977.pdf#search=%22AFLP%20microbial%20fingerprinting%20applied%20biosystems%22.

It is very important to keep the ramp speed around 1deg/sec and to
extend the extension step to at least 1.5min (better 2min). The
touchdown protocol give better results e.g. first 10 cycles with 0.7deg
decrease each cycle.
Also, it is good to include an initial step 72deg for 2min in order Taq
to fill the adaptors and a final step 30min at 60deg. for the
transferase activity of Taq to add a terminal A or T.
I hope this helps

Regards



More information about the Methods mailing list