10.2.3 separation of PCR products and determination of the candidate linkage group of mutation

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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)

1. Prepare 6.2 ml of separation gel mixture (9% Acrylamide) per one gel.
2. After polymerization of separation gel, add 2 ml of stacking gel mixture.
3. A 4.5 mm pitch 18 well sample comb is used for making sample slots.
4. Irradiate fluorescent light to polymerize stacking gel.
5. Prepare 6 acrylamide gels.
6. Assemble electrophoresis apparatus.
7. Fill the buffer chamber with Tris-Glycine buffer (0.0625 M Tris, 0.048 M Glycine)
8. Apply DNA ladder to the first slot of the first gel.
9. 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).
10. 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).
11. Repeat the same thing for the second to sixth gels.
12. Run the gels at 300 V for 50 minutes.
13. Stain the gels with ethidium bromide for 5 minutes.
14. 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

1. Compare the PCR band patterns obtained from wild-type and mutant DNA as templates.
2. 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.