5. Position the gel on the mask and place the cover of the vacublot to hold the mask.
6. Apply the vacuum (~75mm Hg) and initiate the transfer process.
7. Pour 5-10ml of depurination solution to cover the gel and let the solution permeate the gel.
8. Remove the depurination solution and replace it with 20ml denaturation solution and let the solution permeate through the gel.
9. Replace the denaturation solution with neutralization solution and let the solution permeate through gel.
10. Replace the neutralization solution with 10x SSC buffer and let the solution permeate through gel for 30-40mins.
11. Remove excess buffer and turn off the vacuum.
12. Remove the gel and check the transfer under UV.
13. Store the wrapped membrane at room temperature.
Figure 19.3: Step up for vacuum Transfer of DNA from gel to the nitrocellulose membrane.
D. Preparation of labeled probe:
1. Preparation of radioactive probe. There are two different method used to label a single stranded DNA probe either at terminal or throught the sequence.
A. Random primer method- In this method, a random primer is used to anneal to the template and then a PCR reaction is performed in the presence of radiolabeled nucleotide. After PCR, newly synthesized DNA strand is labeled with radioactive nucleotide. The whole process is given in Figure 19.4, and it has following steps-
1. The source double stranded DNA is denatured to generate the single stranded DNA template.
2. A random primer is added and allowed it to anneal to the template strand. It will anneal to the random position through out the sequence at multiple places.
3. Primer will anneal to the template strand and now klenow will start the synthesis of new DNA strand.
4. Newly synthesized DNA will give short stretches of multiple labeled DNA probes.
B. Terminal transferease- In this method, a terminal transferase enzyme will label the probe at the ends to the last nucleotide of the probe. Probe is incubated with the labeled nucleotide and terminal transferase enzyme will add the labeled nucleotide at the end. A partial purification with gel filtration column will give labeled primer.