Differential Display :

Helge Weissig helgew at LJCRF.EDU
Wed Apr 20 11:46:02 EST 1994


At 01:01 PM 04/20/94 +0000, Stephen R. Lasky wrote:
>In article <2p1e7f$b9n at mother.usf.edu>, schnetze at chuma.ec.usf.edu. (Joann
>Schnetzer (BIO)) wrote:

[deletion mutation]

>Did you use the DNase kit that Liang's group sells?
             ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
could you please give us some details about this kit, Steven. I'd be very intersted, since my results w/ DDRT-PCR still vary quite a bit.


>                                                     I have used that and
>RQ-DNase with good results.  One of the things that we found when cloning
>RT-PCR products is that the quality of your first strand synthesis is a
>limiting variable.  If you have really good first strand synthesis, it is
>easy to clone out of PCR (using the TA-Cloning techniques) but if your
>first strand isnt great, its very difficult to get what you want.  I
>carried this idea into DD and got much better and more consistent results
>than by just using the RT part of Liang's kits.  I used the Cycle DNA kit
                                                         ^^^^^^^^^^^^^^^^^
this one too, please :)


>to do two rounds of 1st strand synthesis and got clearer bands that came up
>much more quickly.  The trick with the CycleDNA kit (unfortunately) was the
>methyl mercury denaturation of the RNA.  They have now removed the MeHg
>from the kit since it is toxic and OSHA doesnt like it (or so I was told).
>So the cycleDNA kit is the same as everyone else's cDNA kit.  Liang said he
>didn't need to do this, but I couldn't get my DD gels to look like his
>using his kit.  By optimizing the first strand synthesis they look more
>like the advertized gels (still not as many bands though which is probably
>good.)
>

[more deleted]

>***************************************************************
>Stephen R. Lasky, Ph.D.       Brown University/Roger Williams Medical
>Center
>e-mail: Stephen_Lasky at brown.edu         LandLine: 401-456-6572
>~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
>A nuclear war could ruin your whole day.
>***************************************************************


thanx a bunch already :)
cheers,
Helge



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Helge WEISSIG                              e-mail: helgew at ljcrf.edu
La Jolla Cancer Research Foundation        phone: (619) 455 6480 x253
10901 N. Torrey Pines Rd.                  FAX: (619) 455 0181
La Jolla, CA 92037
U.S.A.

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