yet more cycle sequencing

skspoidn at reading.ac.uk skspoidn at reading.ac.uk
Mon Jun 14 07:51:42 EST 1993


In Reply to  Dan Mytelka (mytelka at mendel.berkeley.edu):
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Anneal: 0.5 pm template and primer (I used 1, 2; my normal standards)
        50 mM Tris 8.8, 7mM MgCl2
        Water to 10ul
      70deg x 3'; 42deg x 10'

Labeling: 5uCi dATP, 1.5 uM other dNTP
          2U Taq
          water to 5.5ul (additional)
      42deg x 5' (I did it at 37deg, because my primer melts around 45)
      Place at room temp (time unspecified; I did short)

Termination: Add 4 ul to GATC tubes with 20uM each dNTP, 60/800/800/400
              uM of the ddNTP respectively
      Various temperatures x 5' (I did 37deg, 50deg, 68deg)
      Stop with 4ul stop solution.
      Heat 95deg, ice quench, load.
--------

This would have to be the wierdest cycle sequencing protocol I have ever seen,
 I mean, it didnt actually involve any cycling, and looks more like a standard 
sequenase protocol to me. I dont know what your template is, but will it melt 
at 70 deg to let the primer in? If your primer melts at 45, then is a 42 
deg anneal a good idea? try heating to 95 for 1 min then slow cool to Room temp.

For the labelling/termination part it should all be done at once 
cyclically, much like a standard PCR reaction. ie (say) 95 1', 37 1', 72 1'  30 cycles

A number of cycle sequencing protocols and problems have already been posted here, 
and all you have to do is retrieve them from the archives. 

fr'instance gopher merlot.welch.jhu.edu, then go thru Usenet news...
search messages...search bionet articles for cycle sequencing

Mike Poidinger
Dept of Microbkitology
Unkitversity of Readkitng
"what is your main purpose?"..."Why to explode of course"
--Sapient bomb, Dark Star



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