WHITE LAB – SOUTHERN BLOTTING PROTOCOL

 

SOLUTIONS

 

Lysis Buffer

                        0.1 M Tris, pH 8.0

                        0.2 M NaCl

                        5 mM EDTA

                        0.2% w/v SDS

                        200 mg/ml proteinase K

 

TE Buffer

                        10 mM Tris-Cl, pH 7.4

                        1 mM EDTA, pH

 

Denature

 

 

Neutralize

 

 

20X SSC

 

 

Membrane Wash

 

 

 

WHITE LAB – SOUTHERN BLOTTING PROTOCOL

 

GENOMIC DNA EXTRACTION

 

1.         Cut mouse tails, place in tubes, and add 0.5 ml of fresh lysis buffer [0.1M Tris, pH 8.0; 0.2M NaCl; 5mM EDTA; 0.2% w/v SDS; 200 mg/ml proteinase K] to each tube

2.         Digest overnight in 55ºC waterbath

 

3.         Spin tubes for 20 minutes at full speed to pellet tail debris

4.            Remove supernatant (avoid hair & debris) to 24-well plates containing 0.75 isopropanol

            Use wide-orifice tips to avoid shearing the DNA.

5.         Leave plates on rocker for 40+ minutes, until DNA precipitates are visible

6.         Use wide-orifice tips to remove precipitated DNA to tubes containing 0.5 ml 70% EtOH

7.         Spin tubes for 10 minutes at full speed

8.         Use gel loading tips to aspirate pellet – make sure pellet is dry

9.         Add 100-250ml TE to each tube

10.            Incubate overnight in 55ºC waterbath

 

11.        Store at 4ºC

 

DIGESTION OF GENOMIC DNA

 

12.        Aliquot 15ml of genomic DNA into fresh labeled tubes

13.        Make up appropriate restriction enzyme digestion and add to tubes

14.            Incubate overnight in 37ºC waterbath

 

GEL ELECTROPHORESIS

 

15.        Run samples on 0.8% agarose gels at ~150V for approximately 3-4 hours

16.            Denature gels for 45 minutes on rocker in denature solution [1.5M NaCl; 0.5M NaOH]

17.            Neutralize gels for 45-60+ minutes on rocker in neutralizing solution [1M Tris; 1.5M NaCl; pH 7.4]

18.        Set up transfer using 10X SSC and Hybond – transfer overnight

 

19.        Mark well positions on membranes

20.        Cross-link the DNA to the membranes using the UV crosslinker

21.        Stain for 5 minutes in water with a little EtBr

22.        De-stain in water for 20 minutes and check under UV to verify transfer

 

HYBRIDIZATION AND PROBE SYNTHESIS

 

23.        Prehyb [30 ml of 20X SSC; 2.5 ml of 20% SDS; 50 ml of formamide; 5 ml of 100X Denhardts (or 10 ml of 50X); 12.5 ml of H2O; 1 ml of herring sperm] for 4-6 hours in 42ºC shaking waterbath in covered containers – place 1 ml of herring sperm DNA in heatblock for 5 minutes, transfer to ice for 5 minutes, then add to prehyb solution

            Need approximately 100 ml of prehyb to prehyb and probe 4 membranes, 50 for prehyb and 20-30 for hybridization

24.        Make probe according to NEB nick translation kit and purify with Nick columns or Qiagen Nucleotide Removal kit

25.        Place probe on heatblock for 5 minutes; transfer to ice for 5 minutes

26.        Add probe to 20-30 ml of prehyb mixture; mix thoroughly

27.        Add membranes to container with mixture; make sure they aren’t sticking together

28.            Hybridize overnight at 42ºC in shaking waterbath

 

29.        Add membrane wash [2X SSC; 0.5% SDS] to fresh containers

30.            Remove membranes from probe solution and add to wash containers

31.        Wash twice for 20 minutes in 42ºC shaking water bath

32.        Let membranes dry on Whatman paper

33.        Wrap in Saran Wrap and expose overnight on autorad cassette or PhosphorImaging cassette

34.        Decant probe into waste tube or save in falcon tube

 

35.            Develop film