5. Preparation of nitrocellulose membrane for transfer: cut a piece of nylon membrane to the size of agarose gel, mark the nylon membrane by cutting on one corner. Wash the nylon membrane with the double distilled water and then soak the membrane in transfer buffer.

6. Cut 2 pieces of 3mm whatman filter paper slightly bigger size to the gel. Wet the filter paper with the transfer buffer.

7. Setup the blotting tray as given in Figure 20.2. Add 500-800ml transfer buffer (10xSSC) in the transfer tray. On a glass solid support, place a piece of non-woven wiper with the size of the solid support to serve as wick. Dip both ends of the wick in the transfer buffer. Wet the wick and remove the trapped air bubble by rolling glass rod.

8. Place the gel on the top of wick and remove bubbles by rolling glass rod.

9. Over-lay nylon membrane on the gel and carefully remove the trapped air bubble by rolling glass rod. (gel and nylon membrane should be handled with wearing gloves).

10. Carefully lay the filter paper on the membrane, remove the trapped air bubble by rolling glass rod. Cover the whole setup with saran wrap to avoid loss of untargeted loss of water.

11. Place a stack of paper towel over the filter paper. Place the glass plate and a weight (0.5-1kg) on the top of glass plate.

12. Allow the capillary transfer for a period of 4-16 hrs. During this period, if filter paper gets wet, change the filter to maintain the capillary action. Do not allow the setup to dry out.

13. Remove the paper towel stack, remove the membrane and rinse with 2xSSC buffer.

14. Check the DNA transfer blotting on the membrane by visualizing the membrane in UV light. Under the UV light, mark the positions of DNA and different lanes.

15. Immobilize the DNA on the membrane with a UV cross linker with an optimal setting.