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*10.2.2 PCR amplification of M-markers from the DNA pools [#uc55b424]

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 [#p32a67b2]
+	Make a genome DNA mixture of 30 mutant embryos (1 ul each, total 30 ul). 
+	Make a genome DNA mixture of 30 wild-type siblings as a control (1 ul each, total 30 ul).
+	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.)
+	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.)
+	Prepare 2 sets of PCR master mix in 1.5 ml tubes (875 ul each; see below).
+	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.
+	Add 4 ul of Ex Taq polymerase (TAKARA) to both tubes. Mix well by pipetting.
+	Dispense 18 ul of the mutant DNA/PCR master mix to the wells in the left (mutant) half of the plate.
+	Dispense 18 ul of the wild-type DNA/PCR master mix to the wells in the right (wild-type) half of the plate.
+	Cover the PCR plate with a silicon rubber lid. Spin down by centrifugation.
+	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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