Jones Lab:Protocols:DNAextraction

DNA Extraction

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1. Add two ball bearings to each 1.2 ml tube containing tissue.
2. Add 300 ul CTAB
3. Disrupt tissue by shaking with paint shaker for 2 min. Repeat if clumps of tissue remain.
4. Centrifuge briefly
5. Loosen lids and heat at 65°C in water bath for 30 min.
6. Let cool to room temperature (15min)
7. Add 300 ul chloroform (done in hood).
8. Shake for 15 sec
9. Centrifuge at 3000 rpm for 15 min.
10. During centrifugation, prepare tubes by adding 200 ul isopropyl alcohol to each tube
11. Transfer 200 μl of the chloroform-extracted supernatant to the new plates. Be very careful not to transfer any of the goop from the interface, or any of the organic layer.
12. Shake for 15 sec
13. Centrifuge at 3250 rpm for 15 min.
14. Pour off the liquid into the sink; the pellet of DNA should stay behind.
15. Wash with 200 μl of 70% ethanol, re-cover the tubes and centrifuge for 7-10 min. at 3250 rpm.
16. Dump off the liquid again
17. Pipette off remaining liquid
18. Air dry (covered in towel) for 10 min
19. Resuspend the pellets in 100 ul of dH2O and let sit for >3 hours at 4°C (I usually leave them overnight).
20. Use 1 μl in a 10 μl PCR reaction.