Competitive PCR - Questions

douglas l feinstein dlfeins at cumc.cornell.edu
Wed Aug 24 17:26:46 EST 1994


Subject: Competitive PCR - Questions
From: Carl Waltenbaugh, cwalten at casbah.acns.nwu.edu
Date: Thu, 18 Aug 1994 18:54:55 -0500
In article <cwalten-180894185455 at 165.124.222.200> Carl Waltenbaugh,
cwalten at casbah.acns.nwu.edu writes:
>	I've been performing competitive RT-PCR on mouse DNA for a while now,
and
>have come up with a unique problem with my Interferon-Gamma analyses. 
When
>I use the primers (published by DNAX) prescribed, I get a ladder-like
>ascension of DNA bands both above AND below my expected products.  Since
>these 'ladders' get in the way of distinguishing competitor product from
RT
>product, I would like to eliminate them if possible.
>
>	Has anyone ever encountered these 'ladders' in their PCR reactions, and
if
>so, what is their origin?
>
>tg
>
>(mistert at merle.acns.nwu.edu)

Subject: Re: Competitive PCR - Questions
From: douglas l feinstein, dlfeins at cumc.cornell.edu
Date: 23 Aug 1994 22:51:43 GMT
In article <33duhv$eu at guru.med.cornell.edu> douglas l feinstein,
dlfeins at cumc.cornell.edu writes:
>Subject: Competitive PCR - Questions
>From: Carl Waltenbaugh, cwalten at casbah.acns.nwu.edu
>Date: Thu, 18 Aug 1994 18:54:55 -0500
>In article <cwalten-180894185455 at 165.124.222.200> Carl Waltenbaugh,
>cwalten at casbah.acns.nwu.edu writes:
>>	I've been performing competitive RT-PCR on mouse DNA for a while now,
>and
>>have come up with a unique problem with my Interferon-Gamma analyses. 
>When
>>I use the primers (published by DNAX) prescribed, I get a ladder-like
>>ascension of DNA bands both above AND below my expected products.  Since
>>these 'ladders' get in the way of distinguishing competitor product from
>RT
>>product, I would like to eliminate them if possible.
>>
>>	Has anyone ever encountered these 'ladders' in their PCR reactions, and
>if
>>so, what is their origin?
>>
>>tg
>>
>>(mistert at merle.acns.nwu.edu)
>
>Somtimes it is a hybrid formed between the true cDNA product and the
>competitive standard.  Changing Mg concentration may help. Make sure your
>samples are cooled , allowing all hybrids to form nicely, before running
>gels.

Subject: Re: Competitive PCR - Questions
From: szcooley
Date: Wed, 24 Aug 1994 16:20:21 GMT
In article <Cv1s1x.7pr at ucdavis.edu> , szcooley at chip.ucdavis.edu writes:
>douglas l feinstein (dlfeins at cumc.cornell.edu) wrote:
>: Subject: Competitive PCR - Questions
>: From: Carl Waltenbaugh, cwalten at casbah.acns.nwu.edu
>: Date: Thu, 18 Aug 1994 18:54:55 -0500
>: In article <cwalten-180894185455 at 165.124.222.200> Carl Waltenbaugh,
>: cwalten at casbah.acns.nwu.edu writes:
>: >	I've been performing competitive RT-PCR on mouse DNA for a while now,
>: and
>: >have come up with a unique problem with my Interferon-Gamma analyses. 
>: When
>: >I use the primers (published by DNAX) prescribed, I get a ladder-like
>: >ascension of DNA bands both above AND below my expected products. 
Since
>: >these 'ladders' get in the way of distinguishing competitor product
from
>: RT
>: >product, I would like to eliminate them if possible.
>: >
>: >	Has anyone ever encountered these 'ladders' in their PCR reactions,
and
>: if
>: >so, what is their origin?
>: >
>: >tg
>: >
>: >(mistert at merle.acns.nwu.edu)
>
>: Somtimes it is a hybrid formed between the true cDNA product and the
>: competitive standard.  Changing Mg concentration may help. Make sure
your
>: samples are cooled , allowing all hybrids to form nicely, before
running
>: gels.
>
>
>I don't understand! How could a hybrid between the cDNA and the 
>competitive standard produce a ladder?
>
>
>#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%
>%                                                        #
>#   Dr. Michael Cooley         Practice Random Kindness  %
>%   mbcooley at ucdavis.edu    and Senseless Acts of Beauty #
>#                                                        %
>%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#
>

Well... I didn't really know the 'extent' of the ladder. We sometimes get
a strong band between competitor and cDNA products. And sometimes a
second minor band a bit higher than the cDNA band. However, these tend to
'come and go' with storage, so I assume they are unstable hybrid molecules



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