PRE: PCR enzymes

PC386 at IL.AC.TAU.VE PC386 at IL.AC.TAU.VE
Thu Jun 25 05:59:54 EST 1992


Hi netters.   On a recent query :

>X-Article-Number: bionet.molbio.methds-reagnts Msg # 374

>I am shopping for PCR enzymes.  I have received a lot of literature
>from various companies telling me all about errors rates, process.
>etc.  I posted a query earlier on this network and received some
>very useful info along these lines.  I plan on using PCR to
>amplify cDNA and then cloning the product for sequencing.  I have
>some enzyme which I can use, it is called Pyrostase from Molec.
>Genetic Resources.  Does anyone have any experience with this
>enzyme?   Are there any enzymes I should stay away from (such
>as Vent, which I have been told leaves ragged ends which are un-
>suitable for RE digest and cloning)?  Any helpful hints will be
>appreciated.

I am using Pyrostase sucssesfully for some time now. Compared with
Taq poly. from HT Biotechnologies (U,.K.) which worked quite allright,
Pyrostase's sensitivity is *MUCH* higher. At the begining, this has
caused some problems, since I didn't realize the great sensitivity.
The optimal concentration for work in my system is 0.5 - 0.75 Unit/Rxn.
(100 ul standard).
More important, the template's concentration with this enzyme MUST be
minimal. When amplifying Genomic DNA in a search of a transgene, I have
found that more than 100ng template/Rxn already cause non specific
bands to appear.
MGR state that the pH is critical, and that a 0.2 unit shift in pH already
reduces activity. I have tested that in my hands and found it to be truth.
- The buffer being supplied by MGR containes (Oddly I think...) BSA.
This is rather tasteless, since BSA frozen in salt containing buffers
tends (and does) to complex to an insoluble product. Note to add extra BSA.

Hope this helps :-)
Good luck, Benny.
 **************************************************************
* Benny Shomer                                                 *
* Tel-Aviv University                                          *
* Sackler School of Medicine, Dept.of Embryology and Teratology*
*--------------------------------------------------------------*
* Snail:  Sheba Medical Center, Tel-Hashomer, 52625,   Israel. *
* E-mail :  PC386 at VE.TAU.AC.IL                                 *
* Tel :  972-3-640-9238     FAX :   972-3-642-2046             *
*                                   (c/o Dr. R. Ehrlich)       *
*                                                              *
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