Long PCR

[Chris Kyle]

I am interested in doing long PCR on the mtDNA that I have isolated from
several species of the marine snail, Littorina. If anyone has any
helpful hints as to possible protocols and/or PCR conditions that I
should be using, I would greatly appreciate the advice. I am hoping to
be to be able to get fragments from 4-8Kb.

Thanks,

Chris Kyle
University of Guelph
Guelph, Ontario
Canada

[Dr. Duncan Clark]

In article <4f8830$6...@ccshst05.cs.uoguelph.ca>, Chris Kyle
<ck...@uoguelph.ca> writes

>I am interested in doing long PCR on the mtDNA that I have isolated from
>several species of the marine snail, Littorina. If anyone has any
>helpful hints as to possible protocols and/or PCR conditions that I
>should be using, I would greatly appreciate the advice. I am hoping to
>be to be able to get fragments from 4-8Kb.
>

Sorry to be picky but why does everyone insist that 4kb is long PCR,
20kb yes but 4kb is well within normal Taq pol PCR.

The Boehringer and Perkin Elmer long PCR mixes work well in our hands to
at least 20kb so yopur 4-8 should be no problem. Keep the denaturation
time low ie 10 secs and the temp down to 92 or 93C. Extend at 68C not
72C.

Duncan
--------------------------------------------------------------------------------
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....
http://www.thenet.co.uk/~dnamp

[Robert Horton]

Chris Kyle (ck...@uoguelph.ca) wrote:
: I am interested in doing long PCR on the mtDNA that I have isolated from

: several species of the marine snail, Littorina. If anyone has any
: helpful hints as to possible protocols and/or PCR conditions that I
: should be using, I would greatly appreciate the advice. I am hoping to
: be to be able to get fragments from 4-8Kb.

Methds-reagnts very own Paul Hengen covered this topic in his August
1994 TIBS column. The article includes valuable comments from Wayne
Barnes himself. It is available at:

ftp://ftp.ncifcrf.gov/pub/methods/TIBS/aug94.txt

so you don't even have to go to the library.

---
Robert M. Horton
http://134.84.47.3 Have a :) day

"Scotty, try flushing the radioactive waste into the ventilation system!"

[Constantin Polychronakos]

> "Dr. Duncan Clark" <dun...@genesys.demon.co.uk> writes:
> In article <4f8830$6...@ccshst05.cs.uoguelph.ca>, Chris Kyle
> <ck...@uoguelph.ca> writes

> >I am interested in doing long PCR ... ..... I am hoping to

> >be to be able to get fragments from 4-8Kb.
> >
>

> Sorry to be picky but why does everyone insist that 4kb is long PCR,
> 20kb yes but 4kb is well within normal Taq pol PCR.

I have to agree with this. My summer student intended to amplify 600 bp of
cDNA by RT-PCR, but used genomic DNA as sample by mistake. We got a gorgeous 4.6
kb fragment that allowed us to clone and sequene two introns!

C.P.