Materials and recipes

Introduction

The Southern blot, invented by EM Southern, 1975, is a means of permanently immobilizing single-stranded nucleic acids to a solid support. The DNA is usually separated by fragment size class by argarose gel electrophoresis. After Southern blotting the DNA is usually detected by "probing" with a complementary DNA probe. This procedure is the Southern hybridation and is  followed a probe detection procedure.

The Southern blot, invented by EM Southern, 1975, is a means of permanently immobilizing single-stranded nucleic acids to a solid support. The DNA is usually separated by fragment size class by argarose gel electrophoresis. After Southern blotting the DNA is usually detected by "probing" with a complementary DNA probe. This procedure is the Southern hybridation and is  followed a probe detection procedure.

To begin this exercise the student needs to have prepared DNA, subjected it to restriction digestion and separated the fragments by gel electrophresis. This series of experiments is outlined on the flow page.

We are doing the third and the fourth steps today.

Last week.

 

1.  Purification of plasmid DNA from a cleared lysate, using the spin protocol of Wizard Plasmid Miniprep.

2.  Digestion of plasmid DNA with restriction digestion enzymes to release the inserted gene.

This week.

3.  Agarose gel electrophoresis and Southern blotting of DNA.

4.  Southern hybridization to identify the gene of interest.

Read: Roche Dig-detection protocol

The first two steps were done last session.

Protocol

wear gloves

1. Cut nylon filters to the same size as the gel. Do not touch the surface of the filter. Wear goves. Handle the nylon with forceps.

2. Soak the nylon in deionized water for 10 minutes.

              Soak the gel in 0.4 M NaOH for 10 minutes.

 

                In the meanwhile, cut filter paer and paper

                towels needed below in steps 3 & 6.

 

3. Examine the blotting rig and ask the instructor about its construction. Pick up the gel from the solution with a piece of Whatmann filter paper that is slightly larger than the gel, whetting the paper on the way. Place the gel face-up on the blotting rig.  The blotting rig contains 0.4 M NaOH (caustic!!); and its construction will be explained by the instructor. See the illustration below.

4. Place the wetted nylon on the gel and smooth out bubbles with a gloved finger.

5. Place plastic wrap over the gel and the whole gel rig. Cut a whole around the gel to expose just the gel, exactly.

6. Place a piece of filter paper on the nylon filter and then a two inch stack of paper towels on top of the whole pile.

7. Let the transfer proceed for one to two hours, four hours at the most.

8. Remove the filter by inverting the filter and gel in register on the blotting rig. Marking the MM markers with a pencil from the gel to the filter, peel off the filter and rinse the filter in 2 x SSC. Do not rub the filter when rinsing. UV cross-link or air dry the filter.

 

The filter is ready for prehybridization.  Put it in a plastic hybridization bag immediately and freeze if necessary. Otherwise go onto prehybrydization.