Differential Display :

Helge Weissig helgew at LJCRF.EDU
Thu Apr 21 12:11:50 EST 1994

The following is the response from Stephen, he asked me to post it:

>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.

The name of Liang's company is GeneHunter Corporation, 50 Boylston Street,
Brookline, MA 02146.  Phone: 617-739-6771, Fax: 617-734-5482.  The DNase
kit is called MessageClean  Kit Cat # M601.  They claim it is superior to
other RNase Free DNases, but I think that RQ1 DNase works as well.

>>                                                     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 :)

Well, as I said below, Invitrogen's cDNA Cycle Kit was the best cDNA
synthesis kit I had found but they took out the MeHg.  I believe that they
will give you the recipe on how to make their new kit into the old kit if
you ask.  Their # is 1-800-955-6288.

>>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

>thanx a bunch already :)

>     God invented surfing, so that the really talented people
>     would not rule the world. - unknown

BTW, as a very old surfer (well, I'm not that old, but I started surfing in
1961) I like your signature.

Stephen R. Lasky Ph.D.  Brown University/Roger Williams Medical Center
LandLine: 401-456-6572                          e-mail: Stephen_Lasky at brown.edu
A booming voice says, "wrong, cretin!", and you notice that you have turned
into a pile of dust.

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