[박상규] Isolation of total RNA from cell line

Isolation of total RNA from cell line

 

 

Materials

-Trizol : cat# 15596-026(invitrogen)

-DEPC treated & autoclaved water(0.2% DEPC)

-Autoclaved blue & yellow tip & eppendorf tube

-Isopropanol, cold 70% ethanol

-PBS

 

1.     Wash the cells in dish with PBS 2 times & remove PBS completely

2.     Add 0.8ml Trizol(6well dish) per well & shake for 20 min & up-down using pippete

3.     transfer the solution to the clean & sterile eppendorf tube

4.     Add chloroform (200ul/1ml Trizol) and vortex for 10 sec.

5.     Centrifuge 12,000Xg at 4 degree for15 min

6.     Take the clean supernatant (450-500ul) carefully and toss to the another clean eppendorf tube

7.     Add isopropanol with the same volume of sample and invert for a while

8.     Centrifuge as like step 5

9.     Discard the supernatant and Add 500ul of cold 70% ethanol

10.  Centrifuge at 22,000Xg at 4 degree for 10 min

11.  Discard the sup and dry the tube

12.  Add 50 ul of DEPC treated water and resuspend the total RNA

13.  Read the observance at 260 nm/280 nm

14.  Now, you are ready to do work using this RNA