SOUTHERN BLOT |
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Last Update: December 2006 |
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This protocol is based on the protocol by Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual, Ed 2. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY |
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| PREPARE SOLUTIONS | |
| 1. 5M NaCl (1 L): | Mix 292.2 g of NaCl with 800 mL of dH2O. Add dH2O to 1 L (Autoclave) |
| 2. 5 M NaOH (1 L): | Mix 200 g of NaOH and dH2O to 1 L |
| 3. 1.5 M NaCl, 0.5 M NaOH (1 L): | Mix 300 mL of 5M NaCl, 100 mL of 5M NaOH, and 600 mL of dH2O |
| 4. 3 M Tris-HCl pH 7.4 (1 L): | Mix 363.3 g of Tris base
with 600 mL dH2O. pH to 7.4 (cHCl ~70 mL). Add dH2O to 1 L (Autoclave) |
| 5. 1 M Tris-HCl pH 7.4, 1.5 M NaCl (1 L): | Mix 333.3 mL of 3M Tris-HCl pH 7.4, 300 mL of 5M NaCl, and dH2O to 1 L |
| 6. 20X SSC (1 L): | Mix 175.3 g of NaCl, 88.2 g of sodium citrate, and 800 mL dH2O. pH to 7.0 with 10N NaOH. Add dH2O to 1 L (Autoclave) |
| PROCEDURE - Membrane preparation | |
| 1. Digest DNA with appropiate enzymes | |
| 2. Separate fragments on a 0.6-1.2 % agarose gel | |
| 3. After electrophoresis, denature DNA by soaking for 30-45 mins in >2 volumes of 1.5 M NaCl, 0.5 M NaOH (constant and gentle agitation) | |
4. Rinse briefly with dH2O and then neutralize with 1 M Tris-HCl pH 7.4, 1.5 M NaCl (20-30 mins with constant and gentle agitation) |
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| 5. Set up a Southern transfer by placing 2 sheet of 3 MM paper (previously wet with 10 X SSC) on top of a support inside a container full of 10X SSC. Place the gel on top of the 3 MM papers (top face down) | |
| 6. Place a sheet of nitrocellulose (previously wet with dH2O, 5 mins, and then with 10 X SSC, 5 mins) on top of the gel, followed by two more 3 MM papers (previously wet with 10 X SSC). Make sure there are no air bubbles trapped between any of these papers | |
| 7. Place a stack of paper towels on top of the 3 MM papers and place a glass support of top of the paper towels with a support (~1 kg) | |
| The objective is to set up a flow of buffer from the container, through the gel, through the nitrocellulose and into the paper towels. Anything that enhances this process will make the Southern work better (like making sure that the buffer is going through the gel and to the nitrocelloluse, and not around the sides) | |
8. After the transfer is complete (8-24 hours), autocrosslink the DNA to the membrane and store dry for later use |
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| PROCEDURE - Southern hybridization | |
| For Southern hybridization, followed the steps described in: cDNA library screening (144) | |