Solutions for Southern Blotting

From ActinoBase

Stock Solutions to Prepare in Advance

Solution Constituents Notes
For DNA transfer Depurination solution 19 ml 13M HCl in 1L water Add acid to water!
Denaturation solution 87 g NaCl, 20 g NaOH in 1L water
Neutralisation solution 77.8 g ammonium acetate in 1L water
20x SSC 175.3 g NaCl, 88.2 g sodium citrate in 1L water Adjust pH to 7 with HCl. Autoclave
For probe labelling 0.2 M EDTA Adjust pH to 8 with NaOH
For Pre-hybridisation and hybridisation 10% SDS 10 g SDS in 100 ml water Heat to 68°C to help dissolve. Adjust pH to 7.2 with a few drops of conc HCl
10% N-laurylsarcosine 5 g in 50 ml water
Maleic acid buffer 22.2 g maleic acid, 17.6 g NaCl in 1L water Adjust pH to 7.5 with NaOH (requires lots, so start off using solid NaOH)
10% Blocking solution 5 g blocking reagent in 50 ml maleic acid buffer Autoclave to dissolve. Solution remains opaque. Can be stored at 4°C for a few weeks.
For detection Detection buffer 12.1 g Tris-HCl, 5.85 g NaCl in 1L water Adjust pH to 9.5

Solutions to Prepare Just Before Use

Solution Constituents Notes
For transfer 2x SSC 20x SSC diluted 10x in water For 1 L: 900 ml water, 100 ml 20 x SSC (Can be stored long term at RT if necessary)
For Pre-hybridisation and Hybridisation Standard Hybridisation Buffer (SHB) 5x SSC, 0.1% N-laurylcarcosine (w/v), 0.02% SDS (w/v), 1% Blocking solution (v/v) For 40 ml (1 blot), add in this order: 26 ml water, 10 ml 20x SSC, 400 μl 10% N-laurylcarcosine, 80 μl 10% SDS, 4 ml 10% Blocking solution
Stringency washes Stringency Buffer A 2 x SSC, 0.1% SDS In this order: 890 ml water, 100 ml 20x SSC, 10 ml 10% SDS (Can be stored long term at RT if necessary)
Stringency Buffer B 0.2 x SSC, 0.1% SDS In this order: 980 ml water, 10 ml 20x SSC, 10 ml 10% SDS (Can be stored long term at RT if necessary)
For detection Washing buffer 0.3% (w/v) Tween-20 in Maleic acid buffer For 1L: 997 ml Maleic acid buffer, 3 ml Tween-20
1% Blocking solution 10% Blocking solution diluted to 1x Maleic acid buffer For 100 ml: 90 ml Maleic acid buffer, 10 ml 10% Blocking solution
Antibody solution Anti-DIG-AP, diluted 1:5000 in 1% Blocking solution For 30 ml (1 blot): 27 ml maleic acid buffer, 3 ml 10% Blocking solution, 3 μl antibody
Colour-substrate solution NBT/BCIP solution diluted 50x in Detection buffer For 1 blot: 200 µl of NBT/BCIP solution in 10 ml Detection buffer. Keep in the dark!