Biotechnology and Bioinformatics (1+1)

Method

1. Wet a membrane carrying immobilized DNA in 2X SSC

2. Place the membrane in a polythene bag, and add ~1 ml prephybridization solution per

5Cm² of membrane. Incubate 3 hr in hybridization incubation oven with occasional

shaking at 60⁰C

3. Just before the end of the prehybridization incubation, denature probe DNA 10min at 100⁰C. Place in ice.

4. Pour out the prehybridization solution from the polythene bag and replace with half the vol. of prewarmed (60⁰C) hybridization solution. Add denatured probe and incubate with shaking overnight at 60⁰C.

5. Pour out the solution and take out the membrance in a glass tray containing 6X SSc and 0.5% SDS. Keep on shaking the tray for 15min at RT. Change the wash solution after 15min.

1. Replace the wash solution with an equal vol. of 2 X SSC and 0.5% SDS and incubate for 10min at RT. Change the wash solution after 15 min.

7. Carry out 15-min medium stringency wash using 45⁰C 2X SSC and 0.5% SDS.

8. If desired, carry out two 15-min high-stringency washes in 2X SSC and 0.5% SDS at 50⁰C.

9. Pour off the final wash solution and blot excess liquid. Wrap in plastic wrap. Carry out autoradiography by placing an X-ray film over the membrane inside an X-ray film holder with intensifying screen at -70⁰C overnight.