PURIFICATION OF DNA WITH PHENOL:CHLOROFORM : ISOAMYLALCOHOL

 

1)    use 25 ml sample DNA ( f.e. after PCR or plasmid DNA)

2)    add 75ml autoclaved water (freshly opened)

3)    add 100ml PCI in a ratio of 24:25:1 (in 4degree fridge cloning only check date)

4)    Vortex all together for 1 minute

5)    Then spin at 10 000 x g for 5minutes

6)    Take the tube out the centrifuge and put the upper phase into a new tube, keep the lower phase in a labeled separate tube for further use and backup control if your procedure fails!!!

7)    To the tube with the upper phase add 100ml chloroform

8)    Vortex 30 sec.

9)    Spin at 10 000 x g for 2 min

10) Take the upper phase (save the lower phase)  and then perform DNA precipitation with it

 

11) DNA PRECIPITATION:

Add 3M Na- acetate 1/10 Vol of upper phase and

Add 2 1/2 Vol of 200 proof Ethanol, (Mol biology only in the hood)

 

Example:

Your upper phase had a volume of 100ml that means you need 10ml Na acetate and 250ml 2oo proof Ethanol

 

12) incubate on ice for 30 min

13) centrifuge at 12 000 x g at 4 degree Celsius for 30 min

14) remove supernatant (whole liquid save in a special tube)

15) go on with pellet and add 200ml 70% Ethanol to tube

16) centrifuge for 5 min at 12 000 x g at 4 degree celcius

17) remove Ethanol and dry pellet (no liquid is allowed at the end, needs to be totally dry)

18) resuspend in 20 ml autoclaved water

19) dissolve at heat block at 37 degree celcius  for 30 min

20) OD