Download Southern Blot

Southern Blot
before you start:
- it is essential that restriction digests are complete, therefore use 5-fold excess of enzyme,
digest for 2 hours, add another 5-fold excess of enzyme and go for another 2 hours.
- for blots of genomic DNA load approx. 10 ug each lane
- electrophoresis is done in 0.8 % agarose/TAE, in the presence of ethidium bromide
- especially for digests of genomic DNA, run the gels slowly, i.e. o/n at 40 V (large gel)
after electrophoresis:
- be sure to take a picture with fluorescent ruler
- wash the gel 20 min in large volume of 0.25 N HCl
- wash the gel 30 min in large volume of denaturing solution
- wash the gel 2 x 15 min in large volumes of neutralizing solution
- set up the blot:
note: filter papers are wetted in 20 x SSC
nitrocellulose is wetted in water, then in 20 x SSC
2 layers of Whatman 3MM
gel
nitrocellulose
3 layers of Whatman 3MM
be careful to exclude all airbubbles!
- do the transfer o/n in 20 x SSC
- rinse briefly in 2 x SSC
- air dry until just damp
- UV-crosslink
- do the hybridization as usual
Reagents:
20 x SSC
denaturing solution: 1.5 M NaCl
0.5 M NaOH
neutralizing solution: 1.5 M NaCl
0.5 M Tris/HCl pH 7.2
1 mM EDTA