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bhairaja pandey 08-09-2006 03:23 PM

problem with faint bands
 
Hi,

I have a problem with faint bands in AFLP. The bands are clear and strong around the higher marker base pairs (around 400 bp) however, the problem is that in the smaller size are not clear or very faint. I am doing Silver Staining for the developlopment of bands.

bhairaja


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StewJW 08-11-2006 08:17 AM

problem with faint bands
 
I've never done Amplified Restriction Fragment Polymorphism but the
Invitrogen kit (11352010) manual or 10544013 for plants has some
trouble shooting tips:

[Only registered and activated users can see links. Click Here To Register...]
[Only registered and activated users can see links. Click Here To Register...] (for
plants).


chovek69 08-30-2006 01:03 PM

problem with faint bands
 

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
[Only registered and activated users can see links. Click Here To Register...].

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



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