Weissenbach set, PCR conditions?

Patricia Rodriguez-Tome pat at genethon.fr
Mon Mar 1 11:58:06 EST 1993


In article <1993Mar1.112907.1 at kean.ucs.mun.ca>, roger at kean.ucs.mun.ca writes:
|> 
|> 	In the Weissenbach paper in Nature [359(1992)794] it is stated that 
|> all the PCR reactions were carried out under the same conditions. 
|> Unfortunately the conditions were not reported except by reference to 
|> "Methods in Mol Genetics, vol. 1 ,in press".
|> 	Can someone post the PCR conditions used for the Weissenbach set of 
|> dinucleotide repeat polymorphisms?
|> 
|> Roger C. Green,	Faculty of Medicine                      Phone: (709)737-6884
|> Memorial University , St. John's, Newfoundland.          FAX  : (709)737-7010


This information is available at gopher.genethon.fr (Genethon Data,
Gmap, Catalog) and at ftp.genethon.fr (pub/Gmap/Catalog/Introduction)


Here is from the "Introduction" file :

PCR amplifications.

All the microsatellite markers described were studied using the
following standard PCR conditions. These conditions represent a
compromise which is probably remote from optimum for most primer
pairs.
PCR reactions are performed in a total volume of 50 5l, containing 40
ng of genomic DNA, 50 pmol of each primer, 1.25 mM dNTPs and 1 unit of
Taq polymerase. 1X amplification buffer contains 10 mM Tris base pH9,
50 mM KCl, 1.5 mM MgCl2, 0.1% Triton X100 and 0.01% gelatin. The
reactions are performed using a "hot-start" procedureJ: Taq polymerase
is added only after a first denaturation step of 5 minutes at 96"C.
Amplification is carried out during 35 cycles of denaturation (94"C for
40 sec) and annealing (55"C for 30 sec). An elongation step (72"C for 2
min) ends the process after the last annealing.
This "hot-start" procedure permits an efficient primary denaturation
step, and minimizes enzyme degradation. Moreover, addition of Taq
polymerase at a high temperature, considerably reduces mispriming.
Since the amplification products to be obtained are short (90 to 350
base pairs long) and the time interval to raise the temperature from 55
to 94"C (obtained with a ramping rate of 1"C/second) is long enough,
completion of DNA elongation can be achieved without a step at 72"C.
This speeds up the overall amplification.




For more information you can send a mail to alain.vignal at genethon.fr


-- 
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Patricia RODRIGUEZ-TOME       Genethon
Internet : pat at genethon.fr
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