Protocol of cGMP experiment

1. Take 1/4th of pellet from given pellet

2. Make solution A(Methanol : Acetonitrile: water) .

(25 ml of Solution A cab be prepared by 10 ml Methanol, 12 ml Acetonitrile and 5 ml water).

( write ratio of above solvents)

3.Add 100l of solution A to the above pellet.

4.Vortex it, and then incubate for 30 min at -20c.

5.Centrifuge at 4c for 15 min at 13000 rpm.

Supernatant
Collect the supernatant in new eppendorff

Pellet
To the pellet, follow from step 3

6. Repeat the procedure for 3-4 times until 1.5ml of supernatant is obtained.

Note: supernatant should be clean. 7. To the collected supernatant, neutralize with 15% ammonium bicarbonate (pH-7). 8. Now vacuum dry the neutralized supernatant of the pellet. 9.To this add 100l of Buffer A and vortex, centrifuge, take the supernatant and injection volume defined as 20l.