ABI 3500XL fragment analysis problem

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ABI 3500XL fragment analysis problem

Postby Loekes » Oct 30 2016 1:51 pm

Hey all, ive been having this problem for a while but couldn't find literature about it so i decided to seek help on forums.

I am doing a multiplex PCR with FAM/HEX labelled primers on Bacteria and doing a fragment analysis on the resulting fragments on the ABI3500XL.
Most of the time i get clear results, but sometimes i get some smaller peaks that should not be there.

As an example: Bacillus Fragilis is supposed to give one peak of 300 basepairs on the Hex channel.
However it gives one large peak at 300, one smaller one at 294, and an even smaller one at 288, so a 6 BP interval here.
This occurs with the signal of some other species as well. I have scanned the target region for tandem repeats, but there are none :/
Some species i get 1 smaller peak, some species i get 2 smaller peaks.
It cannot be oversaturation as well, because then false peaks would come directly after 300Bp, not before.

Any of you have an idea about whats happening here?

I had the idea of doing a smelt curve analysis on the 3 peaks first to see if its really 3 differently sized fragments or not.
After that i would like to pull them apart somehow and sequence them.
can this be done with a high % agarose gel and a low voltage and then gel extract it?

any help is greatly appreciated! or just sharing ideas is great as well!


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