1. Run the gel as normal. Often for genomic southerns it is desirable to run long gels (18cm) over 4-6hrs.
2. Photograph the gel with a ruler adjacent to the molecular weight markers as a reference.
3. 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 with care and wear protective glasses.
4. Add the gel to 250ml alkali transfer buffer, plus 125ml for each additional gel.
5. Place gel on rocker with buffer solution for 20 mins. NOTE low % agarose gels must be agitated slowly to prevent tearing.
6. Keep remaining 500ml for the transfer tank.
Wear gloves for the following steps
7. Cut 2 pieces of large 3MM paper (wicks) and 2 pieces about 2mm smaller than the gel on each edge 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 toweling about 2mm smaller than the gel. The stack needs to be about 6cm thick.
8. Prewet nylon in DDW, then soak in alkali transfer buffer.
9. Add buffer to transfer tank to the level of the platform. Wet the long wicks in transfer buffer and place in the tank.
