Electrophoresis & Transfer; Dna Hybridisation - Thermo Scientific Shake n Stack 6240 Operating And Maintenance Manual

Electrophoresis & Transfer
Electrophorese and transfer DNA fragments on to positively charged Hybridisation
membrane (Böehringer Mannheim, Cat N° 1209 272). Bake at 120°C for 30 minutes to
fix DNA or UV cross-link.

DNA Hybridisation

Prehybridisation and Hybridisation are carried out in bottles in a Thermo rotisserie oven.
Better results have been obtained in bottles than in bags.
1. Pre-warm Hybridisation bottle containing 50ml 2 x SSC to 68°C.
2. Layer the following into a plastic box containing 50ml 2 x SSC:
One piece of nylon mesh (23cm x 23cm - Thermo Scientific).
a)
b) The membrane, DNA side-up.
c) Two layers of nylon mesh.
d) One piece of 'dummy' Hybridisation membrane covering Hybridisation
membrane below.
e) One piece of nylon mesh.
This procedure prevents high backgrounds. (Mesh and 'dummy' membrane are
reusable after washing in distilled water.)
3. Roll 'sandwich' (ensuring no air bubbles are trapped) with DNA side facing inwards.
4. Place roll in Hybridisation bottle and carefully unroll 'sandwich' again ensuring no
air bubbles are trapped.
5. Tip off 2 x SSC and add 20ml prehybridisation buffer pre-warmed to 68°C.
6. Prehybridise in rotisserie oven for 2 hours at 68°C.
7. Denature 300ng DIG labelled probe. Add to 15ml-prehybridisation buffer heated to
68°C.
8. Tip buffer off prehybridised membrane and add Hybridisation buffer to the bottle.
Hybridise overnight in rotisserie oven at 68°C.
9. Remove Hybridisation buffer and freeze. This can be reused a further five times
after heating to 95°C for 10 minutes.
10. Add 50ml 2 x SSC 0.1% SDS to bottle and roll in opposite direction to release
'sandwich'.
© Thermo Scientific, May 2003. Issue 7
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