Medusa Protocol

Required Reagents:

-CHCl3/IsoamilOH (24:1)

-EtOH 70%

-Proteinase K (20mg/ml)

-SDS 10%

-Phenol pH8.0

-IsopropOH

-NaAc 3M, pH5.2

Medusa gDNA Buffer:

Tris-HCl 1M, pH8.0 100ml

EDTA 0.5M 100ml

dH2O 300ml

1) Use trypsin or cell scraper to detach cells from tissue culture flask (T75). Pellet cells and wash twice with PBS 1X. (Always use 15ml falcon)

2) Resuspend pellet in 10ml of Medusa Buffer. Centrifuge cells 10’@1200rpm 4°C. Remove supnt.

3) Resuspend pellet in 3ml of Medusa Buffer, add 70ml Proteinase-K and 400ml SDS 10%. On rotation wheel over night @37°

4) Add 1.8ml of Phenol and 1.8ml of ChCl3/IsoamilOH. On rotation wheel 10’RT°. Centrifuge 10’@3000rpm 4°C.

5) Transfer supnt. to a new falcon, add an equal volume of ChCl3/IsoamilOH. On rotation wheel 10’RT°. Centrifuge 10’@3000rpm 4°C.

6) Transfer supnt. to a new falcon, add 1/10 volume of NaAc and 1/1 volume of IsopropOH, shake gently (or rot wheel) until DNA is precipitated.

7) Meanwhile prepare for each sample:

-glass pasteur hooks

-falcon with 6ml of 70% EtOH

-2ml Eppendorf with 100-200 H2O

8) Fish DNA medusa with the pasteur hook, submerge in EtOH a few times and dissolve in the 2ml

Eppendorf. To completely dissolve, on rotation wheel over night @4°.