Saturday, January 03, 2009

DNA Extraction

Steps of DNA extraction:
01)Rinse mouth with clean water, then gargle with 10mL saline for 3 mins.
02)Transfer the saline with buccal cells into 15mL falcon tube.
03)Centrifuge at 4000rpm, 5 mins, 37'C.
04)Remove supernatant till last drop.
05)Add in 500uL PBS solution and transfer to a 1.5mL microcentrifuge tube.
06)Centrifuge at 5000rpm, 1 min.
07)Remove supernatant carefully.
08)Add in 300uL Nuclei Lysis Buffer, pipette few times to lyse the bucaal cells.
09)Add in 1.5uL RNAse, and inverse the tube gently few times.
10)Incubate at 37'C for 15 mins.
11)Cool to room temperature.
12)Add in 100uL of Protein Precipitate Solution, and vortex vigorously for 20 secs.
13)Centrifuge at 13000rpm, 5 mins.
14)Transfer the supernatant by using a pipette, into a new 1.5mL microcentrifuge tube contained 300uL of Isopropanol.
15)Inverse the tube gently for 3-5 minutes, until the white mass (DNA pellet) is precipitated enough.
16)Centrifuge at 13000rpm, 5 mins.
17)Remove supernatant, and add in 500uL of 70% Absolute Etanol.
18)Inverse gently for few times to wash the DNA pellet.
19)Centrifuge at 13000rpm, 10 mins.
20)Remove the supernatant and air-dry for 10-15 mins.
21)Add in 50uL DNA rehydration buffer and keep in 4'C.

WAHHAHA...... i rewrote the steps without looking at the protocol...

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