Sequencing PCR prod's

jenkins at aidsun.nibsc.ac.uk jenkins at aidsun.nibsc.ac.uk
Fri Feb 11 12:04:47 EST 1994




REcently there have been comments on Gencleaning/sequencing problems on this board.  
OK, my/our technique is relatively straight forward; here goes

1: Carry out 100ul pcr reaction

2: run 10 ul on  1% agarose gel, blot if required.

3: remove approx 30 ul of remaining template and clean using geneclean II.  Ensure ALL alcohol 
    (new wash) is removed after third stage.  Resuspend in suitable volume (depedant up on dna 
    conc); I usually resuspend in 50ul TE

4: Sequence using 32P and sequenase 2, following normal protocol.  However, I carry out the termination incubation at 50oC and routinely have to adjust labeliing diln (from 1:5 to 1:25) with no obvious difference in DNA conc.  Also, annealing is carried out at bpt for 5' followed by 5' in a Etoh/dry ice bath

If req'd I could find our original reference.

Hope this helps


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              Adrian Jenkins
              jenkins at uk.ac.nibsc.comp

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