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 washes	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!