Solutions for Southern Blotting: Difference between revisions
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Latest revision as of 22:23, 23 April 2020
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! |