1. Calculate the volume of each item:
For 1-2 blots For 4- 6 blots
DNA 100ng 300ng
DNTPs 2.0µl 6.0µl
random primer buffer 5.0µl 15µl
Klenow Fragment (1u/µl) 1.0µl 3.0µl
?Á-dCTP*(10uCi/µl) 1.0µl 3.0µl
add water to 17µl 50µl
2. Mix DNA and autoclaved water in a 1.5ml tube, heat in a 100?æ dry bath for 10 minutes to denature the DNA.
3. Cool the tube on ice for 5 minutes, spin it briefly and place back on ice.
4. Prepare the reaction cocktail including dNTPs, random primer buffer, Klenow Fragment. Aliquot appropriate amount into each tube containing probe DNA.
5. Add desired amout of ?Á-dCTP* to each tube and mix well by gently agitating using pippet.
6. Incubate the labeling mixture at 30?æ for more than 3 hours Pre-hybridization
1. Put the hybridization buffer (stored at 4?æ) in 65?æ water bath in order to get the SDS back into the solution.
2. Mix thoroughly and add different volume of hybridization buffer according to the number of blots. The volume of hybridization buffer varies from 10-20ml for 1-8 blots.
3. Eliminate air bubbles and seal the bag. Put the bag into 65?æ air shaker with shaking and prehybridize for 3 hours or 6 hours (new blots). Probing
1. After labeling, add 500µl denature solution (hybridization buffer without Dextran Sulfate) to each reaction tube.
Uncap the tubes and put in a 100?æ dry bath for denaturation. 2. Cool on ice for 5 minutes. Add the denatured probe to the solution in the bag containing membrane and mix well.
3. Put the bag back into a shaker and hybridize for over 10 hours. Washing and autoradiography
1. When hybridization is completed, take out the hybridization bag and take out the blots with a blunt-end forceps and put it into a box containing enough solution of 0.5?ªSSC, 1% SDS. (1?ªSSC, 1% SDS solution for RFLP)
2. Pour off the solution and add adequate prewarmed 65?æ solution of 0.5?ªSSC, 1% SDS (1?ªSSC, 1% SDS solution for RFLP) washing for 15 minutes at 65?æ shaker with gentle shaking.
3. Lay blots on a piece of filter to absorb the solution on the surface, then wrap it right away
4. Put the wrapped blots against the intensifier screen, and put into a film holdr. Insert a sheet of medical X-ray film between the blot and the intensifier screen in a dark room. Expose the film at -20?æ for an appropriate span of time.(For library screening, generally 18-24 hours of exposion; for RFLP, 3-7 days)
5. Exhibit the hybridization band in developing solution, shake briefly in the water and fix figure in fixing solution
6. After exposion, to reuse of the nylon filter, wash the blots using blot washoff solution in order. 10 minutes for blot washoff solution I (optional); 3 minutes for blot washoff solution II; 20 minutes for blot washoff solution I. Lay blots on a piece of filter to absorb the solution on the surface, seal the blots in the hybridization bag for reuse.