Materials and Instruments.

Methodology:

A. Restriction Digestion of Genomic DNA:

1. Isolate the high weight molecular weight genomic DNA from the cells.

2. Digest 10-20µg of the genomic DNA with EcoRI (2-3 unit for each µg genomic DNA) over-night in the presence of appropriate buffer and BSA (if required).

3. Take out small aliquot from the restriction digestion reaction and check on 0.8% agarose gel. Appearance of smear with visible bands indicate complete digestion of genomic DNA and the suitability of the sample for southern blotting.

B. Seperation of DNA on agarose gel: Agarose gel electrophoresis is a standard gel to resolve the DNA and it is discussed in detail in the lecture Lecture 18. As a standard practice, agarose used for southern blotting is of ultra-pure quality to avoid containments affecting migration of DNA. Observe the gel in UV-transilluminator and record the pattern in a gel documentation system.

C. Blotting of DNA on the nylon membrane: Here I am discussing two most popular methods to transfer the DNA onto the nitrocellulose membrane.

i. Capillary Transfer method: The setup to perform capillary transfer of DNA from the gel to the nitrocellulose membrane is given in the Figure 19.2.

1. Soak the gel in 0.25N HCl for 10mins with shaking at RT. This step depurinate the DNA and facilitates the transfer of larger DNA fragments. This setp can be omitted if the fragmented DNA is of smaller size.

2. Remove the depurination solution. Wash the gel with double distilled water. Submerge the gel into the alkalike solution for 30mins at RT with gentle shaking. Alkalike solution will denature the DNA.

3. Remove the denaturation solution. Wash the gel with double distilled water. Submerge the gel into the neutralization solution for 30mins at RT with gentle shaking. This step will neutralize gel without denaturing DNA.

4. Pour off the neutrization solution and incubate the gel in the 10xSSC buffer for 30mins with gentle shaking.