PROTOCOL 011: DNA ISOLATION FROM CELLS IN CULTURE

(1/2013)

 

REAGENTS

 

Lysis Buffer:

10 mM Tris pH 7.5                               For 200 mls:                2 ml 1 M Tris pH 7.5    

10 mM EDTA                                                                            4 ml 0.5 M EDTA pH 8

10 mM NaCl                                                                             0.4 ml 5 M NaCl

0.5% v/v Sarkosyl                                                                   10 ml 10% w/v N-Lauryl sarcosine Na salt

                                                                                                q.s. 200 ml Water

Š       Proteinase K (10 mg/ml)

Š       Tris-equilibrated Phenol/Chloroform, Chloroform, Isopropanol, Ethanol (100% & 70%)

Š       Flame-sealed Pasteur pipettes

Š       1X TE, pH 8

 

PROCEDURE

 

1.     Grow cells to desired confluency.

 

2.     Rinse cells 2X with PBS. At this point, plate can be frozen at -20° C until needed.

 

3.     To each 100 mm plate, add 3 ml Lysis Buffer and 30 mL proteinase K.

 

4.     Incubate overnight in humid chamber at 55 - 60° C.

 

5.     Pour contents of plate into 15 mL conical-bottom screw-cap polypropylene tube (do not use polystyrene).

 

6.     Add 3 ml of Tris-equilibrated phenol-chloroform.

 

7.     Place on rocker for 5 minutes to mix.

 

8.     Centrifuge 4000 rpm for 5 minutes.

 

9.     Remove upper phase with a wide-bore pipette or a clipped tip – this is the aqueous phase and contains the DNA.

 

10.  Add an equal volume of chloroform and repeat steps 7 – 8.

 

11.  Add an equal volume of isopropanol and mix by inverting several times. You should see a small cloud or ‘fuzz-ball’ of DNA.

 

12.  Spool DNA with a flame-sealed Pasteur pipette and remove from isopropanol solution.

 

13.  Dip pipette with DNA into 70% ethanol several times to remove salts.

 

14.  Dip pipette into 100% ethanol to dehydrate

 

15.  Allow DNA to dry before clipping off end of pipette with diamond cutter into 1X TE

 

16.  Store at 4° C.