Molecular Techniques and Methods

Small Scale Genomic DNA Preparation from
Plant Tissues for Polymerase Chain Reaction

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION





MATERIALS AND SOLUTIONS

Extraction Buffer (1 ml)
50 mM Tris-HCl (pH 8.0) ----------------- 50 ul of 1 M Tris-HCl
200 mM NaCl ----------------------------- 40 ul of 5 M NaCl
0.2 mM EDTA ----------------------------- 0.4 ul of 0.5 M EDTA
0.5% SDS --------------------------------- 50 ul of 10% SDS
Deionized H2O ----------------------------- 860 ul




PROCEDURES

1. Grind young leaf (1 cm2) in a microfuge tube briefly.

2. Add 100 ul Extraction Buffer, 5 ul proteinase K (20 mg/ml) solution and 10 ul RNase A (10 ug/ul) solution.

3. Grind for additional 1 minute.

4. Incubate at 37oC for 30 minutes.

5. Extract with 100 ul Phenol: Chloroform: IAA (25:24:1).

6. Extract with 100 ul Chloroform: IAA (25:24:1).

7. Add 1/10th volume of 3 M Sodium acetate (pH 7.0) and 2 volume of ethanol.

8. Centrifuge for 15-20 sec at 2,500 rpm and 4oC.

9. Wash DNA pellet briefly with 70% ethanol.

10. Air dry DNA brielfy.

11. Resuspend DNA in 50-100 ul TE buffer.

12. Use 1-2 ul for PCR reaction.




NOTES




KIT INFORMATION




REFERENCES


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