|Southern Blot |
- Run the gel as normal. Often for genomic southerns it is desirable to run long gels (18cm) over 4-6hrs.
- Photograph the gel with a ruler adjacent to the molecular weight markers as a reference.
- Alkali transfer buffer = 0.5M NaOH (20g/lt), 1.5M NaCl (87.66 g/lt).
Prepare 1 litre for one gel and another 750ml for each additional gel.
BEWARE This buffer is very dangerous, capable of causing severe eye damage. Use the large volumes involved in this procedure wicare and wear protective glasses. Alternatively, use 0.4M NaOH (freshly made from 10M stock).
- Add the gel to 250ml alkali transfer buffer, plus 125ml for each additional gel.
- Place gel on rocker with buffer solution for 20 mins. NOTE low % agarose gels must be agitated slowly to prevent tearing.
- Keep remaining 500ml for the transfer tank. Wear gloves for the following steps.
- Cut one piece of large 3MM paper (wick) and 3 pieces about 2mm smaller than the gel on each side and one piece of nylon (Hybond N+, Amersham) the same size as the gel.
|Large gel (HFI) ||Medium gel ||Mini gel |
|2 (14 x 19 cm) ||2 (15.2 x 10) ||2 (9 x 5.2) |
|2 (14 x 32cm) ||2 (15.2 x 32) ||2 (18.5 x 5.2) |
Cut a stack of paper towelling about 2mm smaller than the gel. The stack needs to be about 6cm thick.
- Prewet nylon in DDW, then soak in alkali transfer buffer.
- Add buffer to transfer tank to the level of the platform. Wet the long wicks in transfer buffer and place in the tank.
- Place gel on platform and spoon on transfer buffer. Add the Nylon and smooth out any bubbles with the back of your finger.
- Add the 2 slightly smaller 3MM filters, the first prewetted, the second dry.
- Add the stack of paper towels. NOTE it is very important that the edges of the towel do not touch the wicks that the gel is sitting on or the transfer will be " shortcircuited".
- Top the stack with a glass or plastic plate and a weight (a bottle with about 200-300ml H2O is ideal). Too much weight compresses the gel and terminates the transfer early.
- Allow to transfer o/n and then remove the stack carefully. Mark the position of the wells on the filter with a biro (and note position of well #1) before removing the filter. Soak the filter in 0.5M Tris pH 7.5, 1.5M NaCl for 5min. after removal from the gel.
- Add filter to 200ml 2 X SSC and allow to soak without agitation for 5'.
Remove filter and blot dry and bake at 80° for 2hrs.
Prehybridize with 10ml Aqua. hyb. (Reagents), 1% SDS and 100ug/ml boiled herring sperm DNA for 20-30 minutes (cloned DNA southern) to several hrs (genomic southern). Alternatively, hybridize with Church Buffer (Reagents) and 100ug/ml boiled herring sperm DNA.
Boil probe for 3' and add to 3ml of fresh hyb solution/SDS/Herring DNA. Squeeze prehyb from the bag and add probe. Seal, avoiding air bubbles and distribute the probe well. (Can also hybridise in bottles). Incubate for 4-6hr+ (cloned DNA) to 16-20hr (genomic southern). Washes are performed in 2 to 0.2X SSC, 0.1% SDS depending on homology of probe to target.