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 tails and mark ears as follows:

                        no cuts:             1

                        left ear cut:       2

                        right ear cut: 3

                        both ears cut:            4

                        short tail:       5

2.         Place tails in Eppendorf tubes and add 0.5 ml of fresh lysis buffer to each tube

3.         Digest overnight in 55ºC waterbath

 

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

5.         Use wide-orifice tips to place supernatant in 24-well plates with 0.75 ml isopropanol

6.         Place plates on rocker for 40+ minutes, until DNA precipitates are visible

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

8.         Spin tubes for 10 minutes at full speed

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

10.        Add 250ml TE to each tube

11.            Incubate overnight in 55ºC waterbath

 

12.        Store at 4ºC

 

DIGESTION OF GENOMIC DNA

 

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

14.        Make up appropriate digestion mixture

            IRS-1: BamH1

            IRS-2: Spe1     

            STF: EcoR1

                        IGF: HincII

                        IMM/TG: BamH1

15.        Add 40ml of digestion mixture to each tube

16.            Incubate overnight in 37ºC waterbath

 

GEL ELECTROPHORESIS

 

17.        Make 0.8% agarose gels

18.        Add 5ml of DNA dye to tubes containing digested DNA

19.        Spin briefly to mix

20.        Load entire sample into wells

21.        Run at ~150V for approximately 3-4 hours

22.        Cut gels and place in numbered containers – add Denature solution

23.        After 45 minutes on rocker, pour off Denature and add Neutralize solution

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

 

25.        Mark well positions on membranes

26.        Cross-link the DNA to the membranes in the Kahn lab

27.        Stain in water+EtBr for 5 minutes

28.        De-stain in water for 20 minutes

 

HYBRIDIZATION AND PROBE SYNTHESIS

 

29.        Add prehybridization mixture (50 ml per 4 membranes)

30.        Prehyb for 4-6 hours in 42ºC shaking waterbath

31.        Make probe according to NEB nick translation kit

32.        Label ~5 ml of probe DNA

33.        Add appropriate dNTPs, 5 ml of 32-P dCTP, and 1 ml Klenow

34.            Incubate for ½ hour in 37ºC incubator

35.        Add 1 ml of Klenow and place in incubator for another ½ hour

36.        Use Nick column or Qiagen kit to purify probe

37.        Add probe to falcon tube containing ~30-40 ml of prehyb mixture

38.        Boil entire tube for 15 minutes; place on ice for 15 minutes

39.        Add to fresh container and place 4 membranes per container

40.        Mix to ensure membranes are sticking to one another

41.            Hybridize overnight at 42ºC in shaking waterbath

 

42.        Add membrane wash to fresh tupperware

43.            Remove membranes from probe solution and add to wash tupperware

44.        Place wash tupperware in 42ºC shaking bath for 20 minutes

45.        Dump out wash and add more for another 20 minutes

46.            Remove membranes from wash and place on Whatman paper to dry

47.        When dry, place in SaranWrap

48.        Expose overnight on autorad cassette or PhosphorImaging cassette.

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

 

49.            Develop film and score