Villanueva, Laura 1-1
DNA Extraction from Gasoline-Contaminated Sediments
(Boston Harbor)
1. Place15 ml sediment in a 50 ml conical tube.
2. Add 15 ml cold AE buffer (Qiagen) OR 10.5 ml 0.12 M phosphate buffer, pH 8.0
plus 4.5 ml CRSR-Red buffer (Bio 101, Inc. CA) as a stabilizing reagent.
3. Add 0.1 mm Glass beads (#11079101 Biospec Products, Inc.) to make a total of
40 ml, as measured in the tube.
4. Freeze in liquid N2 for 10 minutes
5. Transfer to a 65oC water bath for 10 minutes.
6. Repeat steps 4-5 twice.
7. Vortex horizontally for 10 minutes.
8. Spin for 10 min at 2000 rpm
9. Add 1 volume of chloroform.
10. Vortex.
11. Spin for 10 minutes at 2000 rpm
12. Recover top aqueous phase into a new tube.
13. Add 1 vol. saturated phenol (pH = 6.6) Chloroform: Isoamyl alcohol (25:24:1).
14. Vortex.
15. Spin for 10 minutes at 2000 rpm.
16. Repeat steps 12-15 for 3 to 5 times more.
17. Recover top aqueous phase to a new tube.
18. Precipitate the DNA:
a. Start with around 7.5 ml of aqueous phase
b. Add 2 ul of glycogen (5 mg/ml) per ml of sample, about 15 ul
c. Add 1/10 volume 3 M Sodium acetate pH 5.5, about 0.75 ml
d. Add 2 volumes cold 100% Ethanol.
e. Precipitate 1 h at –20oC.
f. Spin 20 minutes at 4000 rpm.
g. Discard supernatant with pipette.
h. Wash with 2 ml cold 75% ethanol.
i. Invert.
j. Keep at –20oC for 5–10 minutes.
k. Spin 10 minutes at 2000 rpm.
l. Discard supernatant with pipette.
m. Repeat steps h-l.
n. Resuspend in 4 ml 75% ethanol and aliquot into four 2 ml eppendorfs.
o. Spin 20 minutes at 13000 rpm.
p. Discard supernatant.
q. Wash with 75% Ethanol twice.
r. Dry upside down
s. Resuspend in TE or PCR water.
t. Additional purification with Qiagen columns is optional.