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RNAi in HeLa cells

时间:2005-07-18 00:00来源:Scienceboard.net 作者:admin 点击: 621次
Sunday, February 08, 2004

Description
This is a popular procedure, in fact, it could be the most popular mammalian genetic technique out there right now. The only issue is that most labs seem to do it differently with variable success. Here is a protocol for HeLa cells that has worked to knock out at least 10 proteins in my hands in two different labs.

Procedure
1. Plate HeLa cells to a density of 100,000 cells in a single well of a 24 well plate using standard DMEM 10% FBS media. Total media volume should be 500ul.
2. The next day, put 3ul of a 20uM stock of siRNA into an eppendorf tube and add 47ul of OPTIMEM media. In a separate tube, add 3ul of Lipofectamine 2000 to 12ul of OPTIMEM media.
3. Allow to incubate for 7minutes at RT.
4. Combine the two tubes and allow to incubate for 25 minutes at RT.
5. Add 38ul of OPTIMEM to the tube and then immediately pipette 100ul of the mixture into the 24 well of HeLa cells plated from the day before (total volume now 600ul).
6. Next day split cells 1:1 into a well of a 6 well plate by first washing the cells with 500ul of PBS, then adding 120ul of trypsin (put plate in 37C incubator for about 5 minutes to trysinize), then add 380ul of DMEM 10%FBS. Put the entire 500ul of resuspended cells into 1.5 mls already put into the 6 well.
7. The next day repeat the RNAi transfection as described EXACTLY in steps 2-5. Do not correct the volume of DMEM on the cells, this second RNAi is a little weaker than the first.
8. Next day change the media.
9. Next day harvest cells and perform Western blot.

Recipes
-none required.

Supplies
-DMEM (invitrogen, sigma, or wherever)
-OPTIMEM (Invitrogen)
-Lipofectamine 2000 (Invitrogen...key to whole protocol)
-siRNAs (Dharmacon Inc.)

Tips
-sterile tips for RNA manipulation

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