Training Manual Application of Genetics and Biotechnology in ...

CHAPTER 6
MITOCHONDFUAL DSA EXTRACTION AND PCR A~TPLIFICATION OF
MITOCHONDRIAL GENES
M.S.Shekhar and G.Gopikrishna
1.a. Procedure for isolation of mitochondrial DNA.
Mitochondria1 DNA can be extracted from the muscle tissue of shrimps by the
following procedure:
1. Wash the muscle tissues (100 mg) in TE buffer (IOmM Tris-HC1, 1mM EDTA,
pH 8.0).
2. Homogenize in 1.5 ml Eppendorf tube with 500 p1 of homogenization buffer
(30mM Tris-HC1, 30mM EDTA, 15 mM NaCI, pH 7.8) containing 100 yglml
proteinase K.
3. Mix the homogenized solution with two volumes of SDS 1%, NaOH 0.2 N.
4. Store samples in ice for 5 min and gently mix with 1.5 volumes of potassium
acetate (3M potassium, 5M acetate).
5. After incubating in ice for 5 min, centrifuge for 10 min at 12000 X g.
6. Extract the supernatant with phenol by adding equal volume of Tris saturated
phenol. Collect the supernatant after centrifigation for 5 min at 12000 X g.
7. Precipitate the mitochondrial DNA after adding two volumes of ethanol.
8. Store at -20°C for 2 hrs.
9. Pellet the DNA by centrifugation for 5 rnin at 12000 X g.
10. Add 1 ml of ice-cold 70% ethanol. Cap tube and mix by inverting several times.
Spin tubes for 1 minute. Pour off supernatant (be careful not to dump out pellet)
and drain tube on paper towel.
11. Allow tube to dry for -5 minutes. Add 50 ul TE or distilled water to tube. DNA is
ready for use and can be stored indefinitely in the freezer.
1.b. Polymerase chain reaction of mitochondria1 genes:
The PCR reaction mixture contains all 4 cWTPs (200 pM), 30 pmoles
concentration of each primer, 1 unit of Taq polymerase and 1X polymerase buffer
containing 1.5 mM MgC12. The thermal program followed was: 93°C for 1 rnin