automated sequencing and PCR products

Michael Poidinger mike at arbo.microbiol.uwa.edu.au
Fri Jun 17 16:49:13 EST 1994


WRT questions about sequencing PCR products and automated sequencing....


I use an ABI sequencer and ABI PRISM kit, and everything in he protocolt book 
works fine.

I am using a one step RT-PCR reaction, purifying the PCR product and 
sequencing with the same primers. The most important thing is to avoid gel 
purification of the PCR.

Although I understand the spin columns work fine, here is a quick and easy 
procedure for PCR product purification

Remove the PCR material from under the oil to an eppendorf (say 20 ul).

Add an equal volume of 4M NH4 OAc  (say 20 ul)

Add an equal volume of isopropanol  (say 40 ul)

mix, centrifuge for 10 min, wash in 70% EtOH, vacuum dry

voila.  PCR product free of contaminating primers, ready for sequence.

Hope this helps

Mike


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Dr Mike Poidinger                Email  mikep at uniwa.uwa.edu.au
Department of Microbiology              mike at arbo.microbiol.uwa.edu.au
University of Western Australia         skspoidn at rdg.ac.uk
Nedlands, 6009, Australia               mp18 at irix.le.ac.uk

  "Smash the control images, Smash the control machines" - W Burroughs
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