Long RT-PCR

Paul N Hengen pnh at fcs260c2.ncifcrf.gov
Tue Jun 7 10:42:17 EST 1994


 In article <1994Jun2.181333.24923 at emba.uvm.edu>
 brianf at med.uvm.edu (Brian Foley) writes:

>    .... I found that if I used
> more plasmid, it was impossible to find optimal conditions.  They
> all worked fine.  In fact the conditions that give maximum yield of
> PCR product when I use just a few copies of plasmid DNA give a poor
> yield of PCR product if I use 100 nanograms of template DNA to start.

What happens if you use too much DNA? Do you see the typical large amount of
EtBr stained material in the well of your agarose gel and smear of DNA like
that reported by others when trying to amplify large DNA segments with the
KlenTaqLA-16 enzyme mixture of Barnes? I'm trying to figure out why these
people can't get it to work.  What exactly is the material in the wells?
Has anyone had this actually work for them?

>	Now that I know about Wayne Barnes and all the recent 
> developments in PCR amplification of large fragments, I would
> try some of the other tricks, such as using mixes of polymerases.

Has anyone tried other enzyme mixtures like those suggested by Barnes?
These include:

1. Amplitaq/Pfu
2. KlenTaq1/Vent
3. KlenTaq5/Pfu
4. Stoffel/Pfu
5. KlenTaq1/Deep Vent
6. Pfu exo-/Pfu exo+

See page 2218 of this paper:

@article{Barnes1994,
author = "W. M. Barnes",
title = "{PCR} amplification of up to 35-kb {DNA} with high
fidelity and high yield from $\lambda$ bacteriophage templates",
journal = "Proc. Natl. Acad. Sci. USA",
volume = "91",
pages = "2216-2220",
month = "March",
note = "long range PCR",
year = "1994"}

*******************************************************************************
* Paul N. Hengen, Ph.D.                           /--------------------------/*
* National Cancer Institute                       |Internet: pnh at ncifcrf.gov |*
* Laboratory of Mathematical Biology              |   Phone: (301) 846-5581  |*
* Frederick Cancer Research and Development Center|     FAX: (301) 846-5598  |*
* Frederick, Maryland 21702-1201 USA              /--------------------------/*
*******************************************************************************



More information about the Methods mailing list