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PCR of G+C rich Chlamy nuclear DNA

Barbara Randolph Anderson bg_lab at ACPUB.DUKE.EDU
Wed Apr 24 09:33:29 EST 1996


As we have heard that some laboratories have had difficulties amplifying
G+C rich C. reinhardtii nuclear DNA, I am submitting my PCR conditions
using 7-deaza-2'deoxy dGTP that have been optimized in the Boynton/Gillham
laboratory in order to amplify an exon of C. reinhardtii nuclear DNA with a
G+C content of 73%.  These conditions are based on the protocol found in
"PCR with 7-deaza-2'-deoxyguanosine triphosphate", Michael Innis, in :  PCR
Protocols:  A Guide to Methods and Applications (1990, Academic Press) and
the protocol supplied with the rTth DNA Polymerase, XL and XL Buffer II
Pack (Perkin-Elmer N808-0187)).  PCR products ranging in size from 0.17 to
2.6 kb, with G+C contents of 62% to 74% have been amplified using various
primer pairs and the following conditions:

PCR conditions for amplifying G+C rich genomic DNA from C. reinhardtii:

200 micromolar each dATP, dCTP, dTTP, Na or Li salts (Promega or Boehringer)

150 micromolar 7-deaza-2'-deoxy GTP (dc7GTP), Li salt (Boehringer)

50 micromolar dGTP, Na or Li salt (Promega or Boehringer)

1.5 millimolar magnesium acetate (Perkin-Elmer)

1X XL buffer II containing Tricine, potassium acetate, glycerol, DMSO
(Perkin-Elmer)

0.2 micromolar each primer (17- to 19-mers, Tm = 52 - 58 degrees C)

ca. 500 ng genomic miniprep total cell DNA (need to use 10 to 100 times
more genomic template than necessary to amplify chloroplast or
mitochondrial sequences)

total reaction volume = 100 microliters.

add 3 units rTth DNA polymerase,XL (Perkin-Elmer) to reaction mix after
temperature of thermocycler reaches 90 degrees C ("hot start" PCR)

        93 degrees C 3 min, 1 cycle
        93 degrees C 1 min, 47 degrees C 1 min, 72 degrees C 3 min
(extended 1 second/cycle), 35 cycles
        72 degrees C 10 min, 1 cycle


Barbara Randolph-Anderson
DCMB Group, Box 91000
Duke University
Durham, NC  27708-1000
e-mail:  bg_lab@ acpub.duke.edu
Tel:  919-613-8159
FAX:  919-613-8177






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