Sunday, September 21, 2008
SIP week 13-DNA diagnostic and research
Helllloooo to everyone!
For the past one week i've been in DNA diagnostic and research lab.
it was quite interesting over there. I learnt about different kinds of genetic diseases, mainly thalassemia.
In DDRL lab, alot of DNA extraction is done and majority of their samples are blood.
Hence, i did DNA extraction and that is what i'm going to post this week.
Firstly cell lysis has to be done.
1. 3 ml of whole blood has to be added to 9ml of RBC lysis solution.It is inverted to mix well and incubated for 5 mins at room temperature.
2. it should be centrifuged at 2000rcf for 2min to pellet the white blood cells.
3.The RBC lysis supernatent is then poured off and the tube is then inverted on a clean absorbent paper for 10 seconds to drain the residual liquid.
4. The tube is then vortexed vigourously to resuspend the cells in the residual liquid.
5. 3ml of Cell lysis solution is added to resuspend the cells in the tube.
6. To lyse the cells, the tube is vortexed on high speed for 10 seconds.
Next protein precipitation has to be done.
1. 1ml of protein precipitation solution is added to the cell lysate.
2. The tube is vortexed at high speed for 20 seconds to mix the protein precipitation solution uniformly.
3. It is then centrifuged at 2000rcf for 5 min. This is to precipitate the proteins
to create a tight, brown pellet.
Following that DNA precipitation has to be done.
1. The supernatant containing the DNA ( leaving behind the precipitated protein pellet) is being poured into a 15ml centrifuge tube containing 3ml 100% Isopropanol
2. the sample are mixed by inverting until the white threads of DNA are being seen.
3. The tube is centrifuged for 3min at 2000rcf, where the DNA will be visible as a small white pellet.
4. The supernatant is poured off and the tube is drained by inverting the tube on a clean absorbent paper. 70% ethanol is added and inverted several times to wash the DNA.
5. The tube is then centrifuged at 2000rcf and then the ethanol is poured off carefully.
6. The tube is then inverted to drain on a clean absorbent paper, and allowed to air dry for 15 mins.
The last step is DNA hydration.
1. 250 microlitre of DNA hydration solution is then added.
2. Sample could be rehydrated by storing at 65 degree celcius for 1 hr or room temperature overnight.
3. the sample is then transferred to a ependorff tube and stored at 4 degree celcius.
This is the whole process of DNA extraction from blood.Its quite technical. If u guys want more explanation, pls do ask me.
Thanks and have fun!
Neela
TG02
