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To: Multiple recipients of list HUM-MOLGEN <HUM-MOLGEN@NIC.SURFNET.NL>
Subject: BIOT: PCR sequencing, RT PCR etc
From: Martin Kennedy <MKENNEDY@chmeds.ac.nz>
Date: Sun, 12 Nov 1995 22:20:29 +1300

This message contains three posts relating to various BIOT
topics, including PCR sequencing and RT PCR:

Martin Kennedy
Arthur bergen
BIOT Topic Editors, HUM-MOLGEN

8<  =======================  cut here  ======================= >8
                      RE>BIOT: various                             =

I can't immediately offer any help but BioTechniques magazine might.  =
They offer a Online Library of their techniques journal.  I know that in =
the past their have been articles which might be just what you're looking =
for.  Their Telnet address is BIOTECHNET.COM with a username BIOTECH and =
a password TECHNIQUE.  If someone else from the list doesn't come up with =
anything, try them OR I also belong to a list that does forensic work and =
I bet that they would be helpful.  I will forward your request on to them =
if you want.
Good Luck!

Melissa Caimano
University of AL at Birmingham
Dept. of Microbiology

8<  =======================  cut here  ======================= >8

Subj:   Re: PCR Sequencing
        Reply to:   RE>PCR Sequencing

Re: PCR Sequencing
We routinely use the Sequenase PCR Product Sequencing Kit from USB.  This =
kit takes dsPCR products and treats them with Exonuclease III and Shrimp =
Alkaline Phosphatase.  The treatment only takes about 1 hour total time =
then you are ready for normal dideoxy sequencing.  The kit costs about =
$300 or so but comes with nucleotides, ddNTPs, all buffers and enzymes =
excluding Taq.  We have had good results with the kit but it has what =
appears to be a limitation, at least in our hands, in that the reads tend =
to be shorter so I wouldn't recommend this method if you want to try to =
read over 275-300 bp per primer/template.  If you would like to know more =
about this kit or other methods that do not involve kits that I have =
used, don't hesitate to call or e-mail.  I would be more than happy to =
help in any way.
Melissa Caimano
Univ. of Alabama at Birmingham
Dept. of Microbiology
(205) 934-9530

8<  =======================  cut here  ======================= >8

For Peter,

The Gentra Purescript kit works well for RNA isolation for RT-PCR at
least for BCR-ABL and t(15;17), short transcripts.  We are not sure it is
sufficient for large transcripts (>2 kb).  The cDNA synthesis is most
efficient when primed with random oligos; then add the specific
amplimers before the PCR step.
We are still comparing different RNA kits and/or methods of specimen
delivery (whole blood and marow) to optimize the recovery and RT-PCR
for larger transcripts. Any suggestions would be appreciated.

Barb Zehnbauer

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