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10.2.2 PCR amplification of M-markers from the DNA pools

Tomoko Jindo
Hiroyuki Takeda
University of Tokyo

M-marker2003 primer list is here
http://medaka.lab.nig.ac.jp/mmarker.htm

Kimura, T. et al.(2004) Mech. Dev. 121, 915-932
Naruse, K. et al.(2000) Genetics 154, 1773-1784

10.2.2.1 PCR reaction with M-markers

  1. Make a genome DNA mixture of 30 mutant embryos (1 ul each, total 30 ul).
  2. Make a genome DNA mixture of 30 wild-type siblings as a control (1 ul each, total 30 ul).
  3. Label the left half of a 96-well PCR plate as “mutant”, the right half as “wild-type”. (Columns 1 to 6 are used for mutant, 7 to 12 are used for wild-type control.)
  4. Dispense 2 ?l of each M-marker (5uM stock) to the corresponding wells in the left (mutant) and the right (wild-type) halves. (48 markers are used for the analysis of a total 24 linkage groups.)
  5. Prepare 2 sets of PCR master mix in 1.5 ml tubes (875 ul each; see below).
  6. Add 25 ul of mutant DNA mixture to one of the PCR master mix. Add 25 ul of wild-type DNA mixture to the other PCR master mix.
  7. Add 4 ul of Ex Taq polymerase (TAKARA) to both tubes. Mix well by pipetting.
  8. Dispense 18 ul of the mutant DNA/PCR master mix to the wells in the left (mutant) half of the plate.
  9. Dispense 18 ul of the wild-type DNA/PCR master mix to the wells in the right (wild-type) half of the plate.
  10. Cover the PCR plate with a silicon rubber lid. Spin down by centrifugation.
  11. Run thermocycling.
875 ul PCR master mix (TAKARA Ex Taq) : 
  (10 X) Ex Taq Buffer 100ul 
  (10 X) dNTP Mixture (2.5 mM each) 80ul      in 875 ul DDW
< PCR conditions >
               95C 120 sec.                               1 cycle.
               95C 30 sec., 55C 30 sec., 72C 60 sec.     30 cycle.
               72C 300 sec.                               1 cycle.
                4C keep

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Last-modified: 2019-04-04 (Thu) 14:18:41