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Stock Solutions to Prepare in Advance
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Solution
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Constituents
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Notes
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For DNA transfer
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Depurination solution
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19 ml 13M HCl in 1L water
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Add acid to water!
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Denaturation solution
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87 g NaCl, 20 g NaOH in 1L water
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Neutralisation solution
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77.8 g ammonium acetate in 1L water
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20x SSC
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175.3 g NaCl, 88.2 g sodium citrate in 1L water
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Adjust pH to 7 with HCl. Autoclave
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For probe labelling
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0.2 M EDTA
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Adjust pH to 8 with NaOH
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For Pre-hybridisation and hybridisation
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10% SDS
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10 g SDS in 100 ml water
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Heat to 68°C to help dissolve. Adjust pH to 7.2 with a few drops of conc HCl
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10% N-laurylsarcosine
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5 g in 50 ml water
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Maleic acid buffer
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22.2 g maleic acid, 17.6 g NaCl in 1L water
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Adjust pH to 7.5 with NaOH (requires lots, so start off using solid NaOH)
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10% Blocking solution
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5 g blocking reagent in 50 ml maleic acid buffer
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Autoclave to dissolve. Solution remains opaque. Can be stored at 4°C for a few weeks.
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For detection
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Detection buffer
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12.1 g Tris-HCl, 5.85 g NaCl in 1L water
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Adjust pH to 9.5
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Solutions to Prepare Just Before Use
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Solution
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Constituents
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Notes
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For transfer
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2x SSC
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20x SSC diluted 10x in water
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For 1 L: 900 ml water, 100 ml 20 x SSC (Can be stored long term at RT if necessary)
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For Pre-hybridisation and Hybridisation
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Standard Hybridisation Buffer (SHB)
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5x SSC, 0.1% N-laurylcarcosine (w/v), 0.02% SDS (w/v), 1% Blocking solution (v/v)
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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
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Stringency washes
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Stringency Buffer A
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2 x SSC, 0.1% SDS
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In this order: 890 ml water, 100 ml 20x SSC, 10 ml 10% SDS (Can be stored long term at RT if necessary)
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Stringency Buffer B
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0.2 x SSC, 0.1% SDS
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In this order: 980 ml water, 10 ml 20x SSC, 10 ml 10% SDS (Can be stored long term at RT if necessary)
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For detection
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Washing buffer
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0.3% (w/v) Tween-20 in Maleic acid buffer
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For 1L: 997 ml Maleic acid buffer, 3 ml Tween-20
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1% Blocking solution
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10% Blocking solution diluted to 1x Maleic acid buffer
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For 100 ml: 90 ml Maleic acid buffer, 10 ml 10% Blocking solution
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Antibody solution
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Anti-DIG-AP, diluted 1:5000 in 1% Blocking solution
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For 30 ml (1 blot): 27 ml maleic acid buffer, 3 ml 10% Blocking solution, 3 μl antibody
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Colour-substrate solution
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NBT/BCIP solution diluted 50x in Detection buffer
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For 1 blot: 200 µl of NBT/BCIP solution in 10 ml Detection buffer. Keep in the dark!
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