RT in taq

[Tracy Aquilla]

In Article <70886010wnr at genesys.demon.co.uk>, Duncan at genesys.demon.co.uk
(Duncan Clark) wrote:
>In article: <199409021840.OAA22887 at medlib.iaims.georgetown.edu> 
nmusto01 at gumedlib.dml.georgetown.edu (NEAL MUSTO) writes:
>> 
>> Tracy;
>> In a word "contamination". We experienced a similar thing andit turned
>> out to be a contaminated OLIGO. Some plasmid containing the cDNA we were
>> interested in, and generally used as a positive control, somehow got in.
>> No matter how we trerated our samples the band showed up. Had to throw
>> out our stocks. Look in to that aspect
>> Neal Musto
>> Cell Biology 
>> Georgetown University
>
>Taq, TthHB8 and TetZ DNA polymerases DO have RT activity. This is well 
>documented. Taq has the least activity. Mn2+ stimulates the RT 
>activity, however the one tube system has never proved to be as good as
>using a two enzyme (MuMLV Rt + taq) system.

In my recent experience, this is not the case. In my hands, the one-tube
one-enzyme (Taq with no Mn2+) system seems to work equally as well as the
one-tube two-enzyme (Taq + superscript II) system. I think the amount of RNA
added to the reaction has a drastic effect on this efficiency, though. I
usually add 2 ug of total RNA to a 100 ul RT-PCR, and get excellent results
with Taq alone.



>
>Duncan
>
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>Duncan Clark                        | Internet:    duncan at genesys.demon.co.uk
>G4ELJ                               | Compuserve:  100015.1406 at compuserve.com
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>
Tracy Aquilla, Ph.D.
Molecular Physiology and Biophysics
University of Vermont
aquilla at salus.med.uvm.edu