CuteChlamyPCR of Chlamydomonas Nuclear DNA

From Barbara Randolph-Anderson, bionet.chlamydomonas April 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