Hello fellow sequencers,
I am beginning a project in which I will be sequencing a few thousand
PCR products using an 377 DNA sequencer. Because of the number of PCR
products, I would like to use dye-terminator chemistries and a 96-well
format. I am a little concerned about obtaining a "clean" (no primers,
salts, etc.) PCR product so that I can use the dye-terminator
chemistries without getting interference from the PCR primers and rxn
mix. I was wondering if anyone has some good ideas on how to clean the
PCRs in a 96-well format.(or should I resort to dye-primer chemistries)
Thank You in advance,
Larry Grocki
Lexicon Genetics
<lgrocki at lexgen.com>
Dear Larry and fellow sequencers ,
There is a paper that I would refer anyone to that solves this problem
rather well , I think it was Rosenthal et al from the Journal of DNA
sequencing and mapping 1990 . They used a PEG precipitation mix to remove
the primers , primer dimers , salts , nucleotides and other assorted
nasties from the PCR reactions . It is an extremely rapid procedure that
is idea for automation and only takes about 20 minutes for the whole
plate . The solution is :
0.6 M NaOAc 15ml 1M for final vol 25 ml ( Use pH 5.2 )
6.6mM MgCl 660ul IM for final vol 25 ml
26.2% Peg 8000 6.55g for final vol 25 ml
The procedure is :
Add 1 vol PEG mix ( usually 20ul reaction )
Leave at room temp for 10 mins
Spin 5 mins
Wash twice with ice old 70% EtOH
Dry
Resuspend in 110% original vol QC 10%
The QC results were then used to determine what vol to use in the
sequencing , 5ul for bright samples and 10 ul for light samples , this
was based on the early TAQ teminator kits ( 20ul reactions ) so vol
reduction will also have to be taken into account . I have seen this
method used to go from colonies to sequence data in under 24 hours with
some great results .
If you are going to use Dye primer chemistry try designing M13 tagged
primers so that you dont have to add the expense and hassle of custom Dye
labelled primers . Again the Dye primer route would be fun to program on
the Biomek .
I hope this is of help ,
Yours Sequencingly ,
David Cain
http://www.lark.com
