PCR specificity

Klaus.Matthaei at ANU.EDU.AU Klaus.Matthaei at ANU.EDU.AU
Mon Apr 18 19:00:53 EST 1994


>Hi Amanda,
>
>Perhaps you should check out your RT step. Maybe your working on contaminating
>
>traces of genomic DNA. Sounds wacky but it does happen, and some controls, like
>^^^^^^^^^^^^^^^^^^^^^

>beta-actin, can lead you astray because of the presence of numerous spliced
>
>pseudogenes. Just a thought.
>
>Phil

Hi Netters

We haven't yet found an RNA isolation method that does NOT contain traces
of DNA.  If our primers do not span an intron then we can always amplify
DNA in RTPCR using very high sensitivity amplification (in a capillary
cycler I might add).  This is true for the standard Chomzynski method or
the now more popular and quicker 'single reagent' methods such as Trisolv,
Ultraspec, Rnazol etc.

Comments anyone about DNA contamination.

Cheers, Klaus

PS: I think Amanda's problem is probably too much template and too low an
annealing temperature.  
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Klaus Matthaei
Gene Targeting
The John Curtin School of Medical Research
The Australian National University
PO Box 334, Canberra, ACT 2600, Australia
E-mail: Klaus.Matthaei at anu.edu.au

"I'd rather a full bottle in front of me than a full frontal lobotomy"
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