Figure 18.2: Isolation of mRNA from total cell lysate.

2. Preparation and casting of denaturating agarose gel:

(A) Preparation of RNase Free Water:

1. Dissolve diethylpyrocarbonate (DEPC) in deionized distilled water to a final concentration of 0.1%.

2. Stir this solution for 12hrs at RT and then autoclave to disintegrate DEPC. Store at RT.

(B) Casting of agarose gel:

1. In a flask, add 1g agarose to the 75ml of RNase free water.

2. Heat the solution to melt the agarose and observe the disappearance of agarose flecks.

3. Allow the solution to cool down upto 550C. Inside a fuming hood, add 10ml 10x MOPS buffer and 18ml 37% formaldehyde.

4. Setup the casting tray with comb and pour the gel in fuming hood (Figure 18.3). [Caution: formaldehyde is very toxic and can be easily absorbed through skin, wear gloves and mask].