Crandall Lab

1.    Cut up tissue (~25mg) into small pieces

2.    Place in labeled 2.0mL tubes

3.    Place in the incubator for 30 minutes to remove excess ethanol

4.    Add 180uL buffer ATL

5.    Add 20uL Proteinase K

6.    Mix by vortexing

7.    Incubate @ 55º on cell rotator until tissue is completely lysed (over night if needed) vortexing samples occasionally

8.    Vortex samples 15 seconds

9.    Add 200uL buffer AL, mixing thoroughly by vortex

10.    Add 200uL ethanol (96-100%), mixing thoroughly by vortex

11.    Pipette the mixture into DNeasy spin columns placed in 2ml collection tube

12.    Centrifuge at 8000rpm for 1 min or vacuum

13.    Discard flow-through from collection tube

14.    Place spin column in new collection tube (old collection tubes can be reused if needed)

15.    Add 500ul buffer AW1

16.    Centrifuge at 8000rpm for 1 minute or vacuum

17.    Discard flow-through from collection tube

18.    Place spin column in new collection tube

19.    Add 500ul buffer AW2

20.    Centrifuge at full speed for 3 minutes or vacuum and then spin for 1 minute

21.    Discard flow-through and collection tube (make sure not to have the spin column touch the flow-through, the membrane needs to be dry for the next step.

22.    Place spin column in clean 2.0ml tube

23.    Pipette 100ul buffer AE onto membrane

24.    Incubate at room temp for 1 minute

25.    Centrifuge at 8000rpm for 1 minute

26.    Repeat steps 23-25

1.    Cut up tissue (~25mg) into small pieces

2.    Place in labeled 2.0mL tubes

3.    Placed in vacufuge for 30 min to remove ethanol

4.    Add 800ul Cell Lysis buffer

5.    Add 20uL Proteinase K (20mg/ml)

6.    Vortex samples 15 seconds to mix

7.    Incubate @ 55º until tissue is completely lysed (over night if needed) vortexing samples occasionally

8.    Vortex samples then spin tubes at 13000rpm for 15 minute.

• Undigested debris will be pelleted

9.    Transfer supernatant to new 2.0mL tube without disturbing pellet.

10.    Add 180 uL of 5M NaCl and vortex well.

• Solution will become frothy.

11.    Spin tubes at 13000rpm for 10 minutes.

• Salted out debris will pellet

12.    Transfer supernatant to cryotubes (screw-cap)

13.    Add 420uL ice-cold isopropanol (2-propanol) to supernatant

• Mix slowly by inversion 5-10 times DO NOT VORTEX

• DNA fibers may be seen at this time

14.    Spin tube at 13000 rpm for 15 minutes.

• DNA pellet may be visible

15.    Pour out supernatan

16.    Add 400uL 70% ethanol to wash DNA pellet.

• Wash 20 minutes on cell rotator at room temp

17.    Spin tubes at 13000 rpm for 10 minutes and pour out ethanol carefully! Pellet may be loose.  If pellet is loose pipette ethanol out being careful to not disturb the pellet.

18.    Dry DNA pellet in speed vac on High for 10 minutes.

19.    Resuspend pellet in 1x TE or purified H2O

• If small pellet add ~ 50uL

• If large pellet add ~ 100uL

20.    Let tubes stand at room temp overnight then place in  -20º freezer.