DNA macro-prep
by Zheng Wang
2/22/05
used for AFTOL polypores
1. grind 2 g dry material in liquid nitrogen, transfer the powder to a 15 ml
tube (VWR #20171-010).
2. add 6 ml cold TES (stored in frig)
TES buffer (500 ml):
10X TE buffer pH 8.0 - 50 ml;
NaCl - 4.12 g;
Adjust by distill. H2O till 500 ml
3. add 100 µl lyticase; let sit at room T for 30 minutes
4. add 200 µl proteinaseK (10mg/ml) (very cheap per Wang); place in warm water
bath at 55co for 60 min
5. add 300 µl 20% SDS; keep in warm water bath at 55co for 45 min
6. add 6 ml phenol; gently shake to mix the two phases very well
7. spin 60 minutes at 4000 RPM at 4 Co (this centrifuge on third floor in
common room [need rotar from LaRochelle lab to hold the 15 ml tubes!)
8. transfer the upper aqueous layer to a new 15 ml tube
9. add 6 ml phenol:phloroform; mix well
10.spin 45 minutes at 4000 RPM at 4 Co
11.transfer the upper layer to a new 15 ml tube
12.add 6 ml chloroform
13.spin 30 minutes at 4000 RPM at 4 Co
14.transfer the aqueous layer to a 50 ml tube (Corning #430291); add 2 volume
100% EtOH and 0.1 volume NaAC; precipitate on ice 15-20 min
15.spin 20 minutes at 4000 RPM at 4 Co
16.dump the EtOH and wash the pellet with 70% EtOH
17.air-dry for 30-40 min, then add 600 µl DNA elution buffer or 1x TE buffer
18.transfer the whole volume to a 1.5 ml eppendorf tube
19.add 20 µl RnaseA over night
20.[next morning] add 10µl ProteinaseK; put at 45-55 Co for 15-30 min
21.add 600µl phenol:chloroform and mix well
22.spin using lab-centrifuge at maximum speed for 10 minutes, and transfer the
upper layer to a new eppendorf tube
23.add 600µl chloroform, mix well, and spin 10 minutes at maximum speed;
transfer upper layer to a new eppendorf tube
24.add 2 volume 100% EtOH and add 0.1 volume NaAC; precipitate on ice for 15
min
25.spin at maximum speed for 10 min
26.wash the pellet with 1000 µl 70% EtOH, then air-dry for at least 30 minutes.
27.add 500 µl 1x TE buffer and check the sample on gel