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*10.2.3 Separation of PCR products and Determination of the candidate linkage group of mutation

Tomoko Jindo~
Hiroyuki Takeda~
University of Tokyo

Gel image 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

**10.2.3.1 Separation of PCR products (Native PAGE)

+	Prepare 6.2 ml of separation gel mixture (9% Acrylamide) per one gel.
+	After polymerization of separation gel, add 2 ml of stacking gel mixture.
+	A 4.5 mm pitch 18 well sample comb is used for making sample slots.
+	Irradiate fluorescent light to polymerize stacking gel.
+	Prepare 6 acrylamide gels.
+	Assemble electrophoresis apparatus.
+	Fill the buffer chamber with Tris-Glycine buffer (0.0625 M Tris, 0.048 M Glycine)
+	Apply DNA ladder to the first slot of the first gel.
+	Apply the PCR products from the first column of the left (mutant) half of the plate to alternate slots of the first gel (10 ul each).
+	Apply the PCR products from the first column of the right (wild-type) half of the plate to the remaining slots of the first gel (10 ul each).
+	Repeat the same thing for the second to sixth gels.
+	Run the gels at 300 V for 50 minutes.
+	Stain the gels with ethidium bromide for 5 minutes.
+	Check the gels under a UV illumination and take photographs.

 < Stock solutions for polyacrylamide gel are as follows (Davis, 1964). >
     Solution A: 1N HCl 31.95 ml, Tris 24.4 g, TEMED 0.305 ml in 100 ml DW
     Solution B: Acrylamide 30 g, Bisacrylamide 0.8 g in 100 ml DW
     Solution C: Ammonium persulfate 0.2 g in 100 ml DW
     Solution D: 1N HCl 12 ml, Tris 1.49 g, TEMED 0.115 ml, Acrylamide 6.25 g,Bisacrylamide 1.56 g in 100 ml DW
     Solution E: Riboflavin 2 mg, Ammonium persulfate 0.05 g in 100 ml DW
   Mix A : B : C = 3 : 5 : 8 for separation gel and D : E = 1: 1 for stacking gel.

**10.2.3.2 Determination of the candidate linkage group of mutation

+	Compare the PCR band patterns obtained from wild-type and mutant DNA as templates.
+	Identify M-markers showing the different band pattern between wild-type and mutant. If the mutant locus is linked with a certain M-marker, the southern derived band in a mutant pool is preferentially amplified and thus more intense compared with that in a wild-type pool. If the locus is close enough, only a southern band is visible with a northern band undetectable.