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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.
"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

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

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