PCR of Chlamydomonas Nuclear DNA
From Barbara Randolph-Anderson, bionet.chlamydomonas April 1996
bg_lab@ acpub.duke.edu
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
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