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3.2 Kb PCR Problems

Mike Dalrymple dalrymple at pplros.demon.co.uk
Wed Dec 6 12:22:59 EST 1995


In article <4a0fsu$nkr at itssrv1.ucsf.edu> Bert Gold, bgold at itsa.ucsf.edu
writes:
>Subject: 3.2 Kb PCR Problems
>From: Bert Gold, bgold at itsa.ucsf.edu
>Date: 5 Dec 1995 03:56:14 GMT
>>Netters,
>
>I am trying to PCR about 3 Kb of Rat DNA in an intron region
>which has been fully sequenced.  I designed primers using Eric
>Lander's PRIMER program and checked them for dimers, hairpins,
>etc. using the NBI PRIMER program.  The primers appear OK by
>compu-inspection.
>
>I am using the Boehringer EXPAND KIT (which doesn't really come
>with a positive control template or primers).  I have prepared
>rat DNA from liver and also have two separate preps of NIH/3T3
>mouse DNA which I've been trying to PCR in parallel.
>
>Based on ODs all of the DNAs look beautiful.
>
>I have tried just now, a set of positive control primers
>used on mouse and supplied by a friend, and I am not
>getting ANY SIGNAL (control should be about 2.4 Kb).
>
>Oh, incidentally, yes, I know that the sequences are
>conserved between mouse and rat in the region I am working
>in.
>
>Normally, I would suspect that the POLYMERASE is not functioning;
>but that is counter to intuition of using a fresh, unopened
>and frozen boxette.
>
>It is true that I have needed to adapt conditions to the
>PE 480 machine (the protocols which come with EXPAND are
>provided for the 9600), but that shouldn't make that much
>difference should it???
>
>My conditions are:  95 degrees for 2 mins.
>               
>   Then, 10X        95 degress for 45 secs.
>                    60 degrees for 1 minute
>                    68 degrees for 4 mins.
>
>   Then, 25 X       95 degrees for 45 secs.
>                    60 degrees for 1 minute
>                    68 degrees for 4 minutes plus 20 secs.
>
>   Then             68 degrees for 7 minutes
>   Then             4 degree soak.
>
>
>Any hints as to what variables to vary, netters?
>
>Suggestions welcomed.
>
>
>Bert Gold, Ph.D.
>UCSF Genetics
>
>
A few thoughts in no particular order:
Have you played around with the annealing temperature?
BCL recommend 20s for denaturation and anealing.
4 min extension for 3Kb seems a bit excessive I'd use 2.5 to 3 mins
Do you use thin walled tubes?
Tried "hot start"? although BCL say its not necessary I do it anyway.

I've successfully PCR'd frags from 7 to 17Kbp using this kit with little
or
no deviation from the recommended conditions.

Good Luck
Mike Dalrymple, Ph.D..



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