Protocol S.5

Southern Blotting

This protocol includes instructions for NENSORB (DuPont) purification of random primed probes for southern analysis.

Solutions

NENSORB A

0.1M Tris 7.7 10 ml 1M Tris pH 7.7

1 mM EDTA 200 ml 0.5 M EDTA

up to 100 ml with Q

prior to use add 14 ml TEA/10 ml

20X SSC

175 g NaCl

88.2 g Na Citrate

pH to 7.0 and bring up to 1 liter

Prehybridization/Hybridization Solution

15 ml 20X SSC

1 ml 0.5 M EDTA

5 ml 50X Dehnardt’s

2.5 ml 20% SDS

2 ml boiled HS DNA (3.0 mg/ml)

up to 50 ml with Q

50X Denhardt’s Solution

5 g Ficoll (type 400 Pharmacia)

5 g polyvinylpyrrolidone (Sigma #PVP-40)

5 g BSA (fraction V Sigma)

up to 500 ml with Q

store at -20°C in 50 ml aliquotes

 

 

 

0.4N NaOH

40 ml 10 N NaOH

up to 1 liter with Q

0.3 N HCl

25 ml concentrated HCl

up to 1 liter with Q

1000X Ethidium Bromide

10 mg ethidium bromide

up to 10 ml with Q

store at room temperature

10X TA

330 mM Tris acetate 7.9 33 ml 1M Tris OAc pH 7.9

666 mM Potassium acetate 13.3 ml 5 M KOAc

100 mM Mg acetate 10 ml 1M Mg(OAc)2

1 mg/ml BSA 10 ml 10 mg/ml BSA

5 mM DTT 1 ml 0.5 M DTT

up to 100 ml with Q

Also need: 100 mM spermidine (20X)

 

Procedure

• Digest 50-100 mg chromosomal DNA overnight at 37°C in 200-500 ml total volume in 1X TA + 1X spermidine.

• Phenol/chloroform extract the restriction digest and EtOH precipitate. Wash the pellet and dry at room temperature. Resuspend in 40 ml TE and quantitate 5 ml. Re-digest 40-50 mg in 40 ul for 2-6 hours. Add 4 ml loading dye and run on a 0.8% agarose gel in TBE or TAE (no ethidium bromide).

• Stain with 1X ethidium bromide--approximately 400 ml for 10 minutes. Rinse with water and photograph.

• Soak in 500 ml 0.3N HCl for 15 minutes, repeat and hold in 0.4 N NaOH. Wet the nylon membrane in NaOH solution.

• Set up an overnight transfer.

• Rinse blot with 2X SSC and bake at 80°C for 1 hour. UV treat for 3 minutes. The blot can be stored indefinitely dry.

• Prehybridize for 4 hours to overnight with 50 ml Prehybridization Solution at 65°C with agitation.

• Resuspend random primed probe in 300 ml NENSORB A and purify as follows:

regenerate column with 2 ml MeOH

wash with 2 ml NENSORB A

load sample

wash with 3 ml NENSORB A

wash with 3 ml Q

elute with 1 ml 50% MeOH into 10 5 drop fractions

dry down and resuspend in 1 ml TE

• Boil the probe and inject it into the prehbridization bag and allow to hybridize overnight.

• Wash blot with several changes of 2X SSC 0.1% SDS and several changes of 0.2X SSC 0.1% SDS at 65°C.

• Autoradiograph.