Southern?Blotting

Southern Blotting:

Detection via Chemiluminescence (BM's Genius System)

Modified to include the use of CSPD for chemiluminescent detection! This is the method we are currently using for Southern blots and non-radioactive detection--it works great for us. Most of the procedure is derived from the Boehringer Mannheim manual "The Genius System User's Guide for Filter Hybridization, Version 2.0, catalogue number 101 023, and the product information flyer for disodium 3-(4-methoxyspiro(1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3,3.1.13.7 ]decan}-4-yl) phenyl phosphate, more commonly known as CSPD (Cat. No. 1655 884).

Blot Preparation:

1. Denature DNA in gel by soaking in 1X denaturation buffer 30-60 min.

2. Neutralize by soaking gel in 1X neutralization buffer, 30-60 min.

3. Place gel on plastic wrap, overlay with prewetted (in water) BM positvely charged Nylon membrane (cat no 1209 299, 1209 272, or 1417 240). Other positively charged membranes work but haven't been tried by us.

4. Place a prewetted 3MM filter paper over membrane, stack about 2 inches of paper towels on top with ~1-2 kg weight to squash it down. Blot overnight.

5. Air dry filter, UV crosslink (Stratalinker) and go to prehybridization step.

Prehybridization (high stringency, 50% formamide method):

Place filter in prehybridization solution and incubate, with gentle agitation, at 42 ℃ for 1+ hours.

It's important from this step forward that the blots not be allowed to air dry, and to minimize exposure to air at any time, as high backgrounds will resμlt.

Hybridization:

Add digoxigenen (DIG) labeled probe to fresh prehybridization solution, and incubate 42 ℃, 2-15 hours, with gentle agitation. Pour off and save hybridization solution. This can be reused for additional blots.

Detection:

1. Wash blot in 2X Stringency Wash solution , twice, 5 min. each, at room temperature.

2. Wash blot in 0.5X Stringency Wash solution , twice, 15 min. each, at 65  ℃ (for ds probes, high stringency). (prepare Buffer 2 at this time)

3. Equilibrate blot 1 min. in Wash Buffer , at room temperature.

4. Block membranes in Buffer 2 , 30-60 min., at room temperature.

(prepare anti-DIG antibody-alkaline phosphatase conjugate, 1/5000 to 1/10000 dilution, in Buffer 2)

5. Pour off Buffer 2, replace with Buffer 2/antibody, incubate 30 minutes at room temperature.

6. Pour off Buffer 2/antibody, and wash blot in twice in Wash Buffer , 15 minutes each at room temperature.

7. Equilibrate blot 2 min. in Buffer 3

(prepare CSPD at this time; BM recommends a 1/100 dilution in Buffer 3*, requires 0.5-1.0 ml/100 sq cm.)

Note added 2/16/99 : Frank Verholen has reported that a 1/5000 dilution of the CSPD in Buffer 3 is equally effective; this is currrently being tested in our lab

8. Pour off Buffer 3, quickly place filter between 2 sheets of cellophane (or some sort of plastic sheets--we use cut-up heat seal bags), and drop 0.5 to 1.0 ml of CSPD-Buffer 3 onto filter (DNA side up). Gently spread over entire filter by pressing the top sheet over the filter. Incubate at room temperature and then wipe off the excess liquid. Incubate the sealed blot at 37℃ for 5 to 15 min to enhance the luminescent reaction. Expose X-ray film (we've had great success with Kodak's BioMax MR film ). Exposure time is 30 min-4 hours; light emission intensity increases for 7-8 hours after application of CSPD, and is maximal for about 12 hours.

Probe preparation:

This is the random priming method, for DNA templates of 100-10,000 bp.

Mix the following:

DNA* (10 ng-3 ug) water (to 15 μl) 10X hexanucleotides 2 μl 10X dNTP mix 2 μl Klenow 1 μl

*place in boiling water bath, 5-10 minutes, and ice.

1. Incubate 1-12 hours, 37 ℃.

2. Stop reaction with 2 μl 200 mM EDTA.

3. Add 1 μl glycogen (10 mg/ml stock). Precipitate with 0.1 volumes (2.1 μl) 4 M LiCl and 3 volumes (66 μl) absolute ethanol. Place at -70C 10 minutes, and pellet in microfuge 15 minutes.

4. Wash pellet one time with 70% ethanol, dry. Resuspend DNA in 50 μl TE/SDS.

Reagents:

10X Random primers

62.5 A260 units/ml random hexanucleotides in 500 mM Tris-HCl, 100 mM MgCl₂, 1 mM Dithioerythritol (DTE), 2 mg/ml BSA, pH 7.2 (we use the random primer-buffer from BRL as 10X)

10X dNTP (with digoxigenin-11-dUTP)

1 mM dATP, 1 mM dCTP, 1 mM dGTP, 0.65 mM dTTP, 0.35 mM DIG-dUTP, pH 6.5

(available in the Genius 1 or Genius 2 labeling kits, DIG-dUTP also available separately as 1 mM [cat no 1093 088]and 10 mM [cat no 1209 256] solutions)

TE/SDS

10 mM Tris, pH 7.5

1 mM EDTA

0.1% (w/v) SDS

Prehybridization/Hybridization solution

50% (v/v) formamide

5X SSC

0.1% (w/v) sarkosyl

0.02% (w/v) SDS

1% (w/v) BMB blocking reagent for nucleic acid hybridization

Stringency Wash solutions

2X   0.5X 2X SSC 0.5X SSC 0.1% SDS 0.1% SDS

Genius Buffers (for use with CSPD)