cycle sequencing from mini prep DNA

Euan R. Taylor etaylor at ccu.umanitoba.ca
Thu Apr 29 12:50:13 EST 1993


In <C5yLxL.3I9 at ccu.umanitoba.ca> hamel at ccu.umanitoba.ca (Andre Hamel) writes:


>The alkaline conditions remove ALL RNA, which inhibits sequencing rx'ns, also
>the amount of template generated (usually a few ug) should be sufficient
>for MANY cycle sequencings, otherwise such a prep yields enough ds plasmid
>for no more than three/four regular Sequenase type rx'ns (example, if you
>want to sequence from both ends, from middle, etc).

Do you find RNA that much of a problem? I frqguently dont bother to remove
it from my minipreps, although I do phenol and chloroform extractions, and
it doesn't seem to affect the reaction significantly with the cycle
sequencing procecure, though I always used to remove it to use sequenase
and so on. If I don't phenol extract and re ppt after a simple lithium
chloride lysis and ammonium acetate/isopropanol pptn procedure the salt and
residual gunk screw things up. 
But I never get NO signal at all whichis what I think Joy is having a
problem with. I
always get something, it can just be an unreadable mess if the template is
dirty.
How much DNA do you use in a reaction by the way, I find the recommened 50
fmol gives signals which are way to weak to be useful, I try and use 1/2
to 1ug usually for good rapidly visible ladders.
cheers
euan


-- 
Usual disclaimers. These are my own opinions and probably nobody else's.
etaylor at ccu.umanitoba.ca
"All great truths begin life as blasphemies"  George Bernard Shaw 



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