PCR amplification problems could use some help

Robert Hartley r.hartley at freeuk.com
Tue Jan 4 11:07:12 EST 2000


In message <uUVlQJB6qbc4EA8$@genesys.demon.co.uk>
          "Dr. Duncan Clark" <Duncan at nospam.demon.co.uk> wrote:
Hello Duncan.
Happy Christmas to you and Nelson.
> In article <6239da7a49.root at freeuk.net>, Robert Hartley
> <r.hartley at freeuk.com> writes
> >I see you are using an Ab? 
> >Try this

> >94-5/10mins
> 
> 
> If you use the RapidCycler with an Ab surely you don't do a 10 min step
> first? There is no need. I know of labs using the Ab on LightCyclers and
> they find the Ab to be inactive after just a few seconds or even less at
> 94C. It works very well as a HotStart though.
I was assuming the Ab was for Hotstart. 
I don't know the life of the Ab in a tube but I thought the rapid Ab 
inactivation was due to surface denaturation. Wouls the BSA coat enough to 
prevent this?

The poster was using a PE9700. The 10 mins didn't refer to the Rapidcycler.
:-)
Quote
"The only variables that are different are the technician, location, water, 
and machine. We are currently using the Perkins Elmer 9700 thermocycler." end

As for the variables. 
Technician; Doubt it
Location; ditto
Water; Nuclease free?
Machine; almose certainly if trying to swap over a protocal.


> 
> Duncan

Cheers
Bob (in bed with the flu since 28/12/99) Happy new year. :-))





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