DNA Transfer - Southern Method
1. Following electrophoresis, place gel in 500 mL of:
0.25 M HCL 10 mL HCL
490 mL ddH2O
2. Rinse at 22° with agitation for 20 min, until bromophenol blue turns yellow.
3. Decant solution and rinse chamber with water. Add 500 mL of:
0.5 M NaOH 10 g NaOH
1.0 M NaCl 29 g NaCl
500 mL ddH2O
4. Decant solution and rinse chamber with water. Add 500 mL of:
0.5 M Tris, pH 7.0 30 g Tris Base
1.5 M NaCl 44 g NaCl
900 mL ddH2O
Adjust pH to 7.0 with HCL
5. Fill a tray with 500 mL 4X SSC. Place a glass plate over the tray and drape Whatman 3MM paper over two edges to form a wick.
6. Place gel on Whatman paper wick. Roll out air bubbles with a pipet.
7. Soak Nylon and 1 piece of Whatman 3MM in 4X SSC. Place cut Nylon filter on gel. Clip the upper left-hand corner to orient.
8. Cover the gel and Nylon with wet piece of Whatman 3MM paper.
9. Cover all areas of the gel not to be transferred with plastic wrap.
10. Place a stack of paper towels on to of gel stack. Weigh down slightly.
11. Transfer DNA overnight. Remove filter and air dry.
12. Crosslink DNA by exposure to uv light for 30 sec.
Last Update: Thursday, 19-Jun-2014 11:50:31 PDT
This page has been viewed 428 times.
eckofoid at ucdavis.edu