TaqMan Questions

Dr. Duncan Clark news at hgmp.mrc.ac.uk
Tue Jul 24 05:15:24 EST 2001


In article <3B5C56B4.9DE87B87 at ilw.agrl.ethz.ch>, the eminent Gottfried
Dasen at ETH wrote
>I have two questions concerning the TaqMan PCR technique:
>
>I want to detect the "total bacterial DNA" load of various samples
>(food, ....). (in order to compare it with the incidence of other
>genes).
>Does anybody have a suggestion for a promising target gene (16S rDNA is
>not a single copy gene, so its of no real use to me).
>Ideally such a gene should be ubiquitous (well, I'll settle for: present
>in most bugs), single copy, enough DNA-seqs published, ..
>
>Do you have any suggestions ?

PolA - DNA polymerase I. I have seen a few years ago a paper using
primers against the conserved regions. Possibly a paper detailing
cloning of a thermophilic Bacillus polA or Mycobacterial polA.
 
>
>Since I'm going to quantitate DNA, I  also must use a quantitative DNA
>extraction method.
>So far nobody seems to have payed attention to these fact or at least I
>couldn't find any work on it.
>
>Does anyone have experience with this ?

The best way for quantitative extraction would be to spike your sample
with a standard i.e. phage DNA - lambda(?) or spike with a.n.other
organism possibly archaeal in origin. 

Duncan
-- 
Homogeneous Fluorescent Reporting Systems for Real-Time Quantitative PCR: 

Optimisation, Probe Technology & Future Systems

4-5 September 2001
King Alfred's College, Winchester, UK 

http://www.dera.gov.uk/html/news/events/real_time_quantitative_pcr.htm




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