Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo

How to transfect siRNA into suspension cell lines?


  • Please log in to reply
3 replies to this topic

#1 popogirlxd

popogirlxd

    member

  • Active Members
  • Pip
  • 19 posts
0
Neutral

Posted 01 February 2005 - 06:15 PM

Hi, everyone,
Does anyone can tell me the protocol to transfect SiRNA into suspension cell?Thanks a lot!

By the does any one obsever if siRNA can induce apoptosis?

#2 fred_33

fred_33

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 288 posts
0
Neutral

Posted 02 February 2005 - 02:52 AM

Hi

normally, you can get these protocols on the user maual of your transfection reagent.

Transfecting Suspension Mammalian Cells
Use the following procedure to transfect mammalian cells in suspension in a
6-well format. All amounts and volumes are given on a per well basis.

1. On the day of transfection, prepare a single cell suspension from stock cells.
Wash the cells once with serum-free growth medium without antibiotics,
and seed cells at a density of 2-3 x 106 cells per well in 0.8 ml of serum-free
growth medium without antibiotics.
2. For each transfection sample, prepare complexes as follows:
a. Dilute 1-5 µg of DNA in 100 µl of Opti-MEM® I Reduced Serum Medium
(or other medium) without serum.
b. Mix Lipofectin® before use, then dilute 2-25 µl of Lipofectin® in 100 µl of
Opti-MEM® I Medium (or other medium) without serum. Let stand at
room temperature for 30-45 minutes.
c. Combine the diluted DNA with diluted Lipofectin® (total volume =
200 µl). Mix gently and incubate for 10-15 minutes at room temperature
(solution may appear cloudy).
3. Add the 200 µl of complexes to cells. Mix gently by rocking the plate back
and forth.
4. Incubate cells at 37°C in a CO2 incubator for 5-24 hours.
5. The following day, add 4 ml of complete growth medium to the cells.
6. Incubate cells at 37°C in a CO2 incubator for 24-48 hours prior to testing for
transgene expression.

#3 popogirlxd

popogirlxd

    member

  • Active Members
  • Pip
  • 19 posts
0
Neutral

Posted 02 February 2005 - 06:02 PM

Thank you very much for your reply!

#4 fred_33

fred_33

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 288 posts
0
Neutral

Posted 03 February 2005 - 12:58 AM

hi !

please tell me if you succeed B)




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.