96-well PCR cleanup?

David David
Mon Apr 7 17:22:57 EST 1997


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



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