Lab day 5

u = micro

For approx 100mg of cells :

1. resuspend cell mass in 500 ul of TNE buffer

2. Add 10 ul lysozyme and vortex [very cool thingy!]. Incubate at 37 degrees for 30 min.

3. Add 10 ul potinase K and 20 ul of 10% SDS. Vortex. Incubate this at 55 degrees Celcius

4. Look under microscope and check if cells have lysed. If yes continue protocol, if not pellet and repeat from step 1.



5. Extraction with equal vol of phenol/cholorform/isomylalcohol (25:24:1)

6. Invert a few times and then spin in a centrifuge at 12, 000 rpm fo 5 mins

7. Remove the supernant and place in a fresh eppendof tube

8. repeat 6 and 7 twice. ie. we're spinning the supernant (because it contains the DNA) and getting rid of the pellet (contains the protein crap)


9 Add 1 volume of isopropanol and 1/10 vol of 4M NH40Ac,
invert and place in 4 degrees Celcius over night to allow precipitation er, its friday ...? Leave til Monday? [dunno yet]

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10. centrifuge to collect the DNA at 12, 000 rpm for 10 mins

11. Remove supernant ad wash pellet with 70% EtOH

12. centrifuge for 12. 000 rpm for 10 mins and dry

13. redissolve DNA 100 ul of buffer

14. check for DNA extraction on a 1% agarose gel.