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
- - - - -

complicated transfection requires professional help


  • Please log in to reply
3 replies to this topic

#1 sarigsk

sarigsk

    member

  • Members
  • Pip
  • 4 posts
0
Neutral

Posted 24 May 2009 - 02:12 AM

Hi
I intend to use the dual-luciferase reporter assay for quantitative measurement of gene expression.
I want to produce stably transfected cells that will express both renilla and firefly luciferases under the control of independent promoters and that the cells will also be expressing GFP constitutively (necessary for sorting the cells by FACS).
I badly need help to address two problems:
1) I need an internal control to relate the luciferase expression to that allows stable expression assay (i.e, contains selectable marker and is constitutively expressed)
2) I guess that the excitation of GFP by renilla luciferase might be a problem as the light emitted by renilla reaction is at 480 nm well within the peak for excitation of GFP (470-490 nm). will that actually happen?

Any advice will be highly appreciated
Thanks in advance

#2 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,738 posts
400
Excellent

Posted 24 May 2009 - 04:29 PM

GFP will be excited by the light generated from the renilla reaction, but it may not be detectable as there are usually significant losses in fluorescence. You will need to run a control for this experiment using non-transfected cells, transfected cells that produce the GFP alone, cells that express the renilla luciferase... etc., I am sure you can work out the permutations.

#3 sarigsk

sarigsk

    member

  • Members
  • Pip
  • 4 posts
0
Neutral

Posted 27 May 2009 - 08:10 PM

many thanks bob1 for your reply but what about the 1st question. is there any company that provides vectors like B-gal that can work as an internal control BUT CONTAINS A MAMMALIAN SELECTABLE MARKER? because in my case i am not going to use renilla as an internal control

#4 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,738 posts
400
Excellent

Posted 28 May 2009 - 04:16 PM

Try looking here:'http://plasmid.med.h...LASMID/Home.jsp




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

©1999-2013 Protocol Online, All rights reserved.