10.2.2 PCR amplification of M-markers from the DNA pools †
University of Tokyo
M-marker2003 primer list is here
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 †
- 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.