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Several hybridisation buffers can be used for this procedure and it can be performed in roller bottles, sealable containers or heat-sealed plastic bags. Here in the Blaxter Lab, QuickHyb® is the hybridisation buffer currently used and hybridisation is performed in roller bottles .
1 Prehybridise the membranes by incubating in pre-warmed (65°C) hybridisation buffer, for at least 1hr at 65°C, with rotissery motion. Allow 50-100 microlitres /cm2 of membrane in roller bottles .
2 Label probe DNA using 'rediprime' random primer labelling protocol, add denatured DNA template in a final volume of 45 microlitres ddH2O Flick the tube to mix, Spin briefly Add 5 microlitre of [32P]dCTP, mix by gentle pipetting Spin briefly Incubate at 37°C for 10 minutes Stop reaction by addition of 5 microlitres 0.2M EDTA For hybridisation denature4 labelled DNA Chill on ice prior to use
3. Pre-warm 1ml hybridisation buffer in bijoux tube Add labelled probe DNA to hybridisation buffer Mix gently by pipetting
4 Transfer diluted probe to roller bottle Seal bottle and mix buffer/probe solution by gentle shaking Return roller bottle to rotissery
5 Incubate overnight at 65°C with constant mixing
6 Add 1/3 roller bottle volume of x2 SSC/0.1% SDS Mix Discard wash
7 Add 1/3 roller bottle volume of x2 SSC/0.1% SDS Return to rotissery Incubate for 15min
8 Repeat step 7
9. Check radioactivity with hand held Geiger Counter
10 Add 1/3 roller bottle volume of pre-warmed (65°C) x0.1 SSC/0.1% SDS Return to rotissery Incubate for 15min
11. Repeat step 10, usually about 3 washes until counts over clear membrane areas low, by contrast to activity over colony area
12 Transfer membrane to cling-film sheet: colony side down Sandwich with second cling-film sheet Trim excess cling-film to about 1cm around meembrane Fold excess cling-film back over membrane to seal edges of sandwich
13 Place membranes between sheets of acetate/plastic cut to fit autoradiograph cassette This prevents wrapped membranes adhering to film during exposure
14 Insert autoradiography film
15 Exposure time vary from 4-24hr depending on: [probe], stringency of washing and quality of 32P-radioisotpe
QuikHyb® (Catalog # 201221; Stratagene Ltd., UK)
Rotissery Hybridisation Oven (Hybaid, UK)
If bags are used allow 200 microlitres buffer/cm2 of membrane
'rediprime' random prmer labelling (RPN1633; Amersham International plc, UK)
Denature by heating to 99°C for 5 minutes in PCR block or bioling water-bath
Rediprime tubes can labell up to 25ng template DNA/tube
Hyperfilm™-MP (Amersham, UK)