SOUTHERN BLOT TRANSFER

 

Denaturation of DNA

1. Remove agarose gel from electrophoresis tank and photograph next to a fluorescent ruler. Cut the lower corner off the gel to distinguish it from others.

2. Turn gel over on a glass plate and break DNA with Stratalinker set at 5000 mJoules (if DNA is digested) or at 8500 mJoules (if DNA is non-digested).

3. Soak the gel in Denaturing Solution for 30 min. at RToC with shaking.

4. Soak gel in Neutralizing Solution for 30 min. at RToC with shaking.

5. Cover gel with 20X SSC.

 

Transfer

1. Set up a platform in a glass dish to support the gel.

2. Cut a piece of Whatman 3MM paper for a wick that is wider than the gel and long enough so that both ends reach the bottom of the container when it is resting on the platform, and two smaller pieces the size of the gel, and soak in 20X SSC.

3. Fill the container with 20X SSC (approx. 2 cm) and place the wick over the platform. Wet the wick thoroughly and smooth out all air bubbles with a pipette.

4. Cut a piece of Nytran membrane about 1 mm larger than the gel, label it first, and wet in dH2O then 20X SSC. Cut the corner to match the gel.

5. Place the gel upside down onto the wet wick. Smooth out all air bubbles.

6. Surround the gel with parafilm so that the transfer buffer must flow through the gel and cannot "short circuit" around the gel.

7. Place the wet Nytran membrane on top of the gel & smooth out all air bubbles, then place 2 pieces of Whatman 3MM paper the same size as the gel on top.

8. Place stacks of paper towels on top of the Whatman 3MM paper and place a weight (e.g., Fisher or VWR catalogue) on top. (Note: be sure that neither the membrane, the Whatman 3MM papers, nor the paper towels contact the wick.)

11. Allow transfer to proceed O/N at RT°C (7 hr for a minigel).

 

 

 

End of transfer

1. Remove paper towels and Whatman 3MM paper from Nytran membrane and gel.

2. Mark the location of the gel slots on the membrane with a ball-point pen.

3. Remove and discard the gel.

4. Rinse the Nytran membrane with 2X SSC to get rid of agarose.

5. UV Auto-crosslink the Nytran membrane to fix the DNA (DNA face up).

6. Keep at 4°C in 2X SSC before hybridization.

 

Buffers for Southern Blot

20X SSC

NaCl 175.3 g

Sodium Citrate 88.2 g

Adjust pH to 7.0

Qs. to 1L with dH20

Autoclave

Denaturing Solution (1.5 M NaCl, 0.5 N NaOH)

5 M NaCl 300 ml

10 N NaOH 50 ml

Qs. to 1L with dH20

Neutralizing Solution (1.5 M NaCl, 1 M Tris pH 7.4) 0.5M Tris is also fine

5 M NaCl 300 ml

Tris base 121.1g (or 500ml of 1M Tris pH 7.4)

Qs. to 1L with dH20

Adjust pH to 7.4