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
- - - - -

Transfection problems


  • Please log in to reply
5 replies to this topic

#1 Tigz

Tigz

    member

  • Members
  • Pip
  • 1 posts
0
Neutral

Posted 07 October 2009 - 01:56 AM

Recently I have been having problems carrying out transient transfection of 293T cells using Sigma Calcium Phosphate transfection kits. One day it works and the next day it does not under the exact same conditions. I have tried changing transfection kits, cells, and plasmid DNA. All the effort has failed to shed any light. There will be a day it works and the next it does not. After spending a whole of May trobleshooting this problem I finally got it to work but its stopped working again. I concluded that it must be the state of the cells because the plasmid DNA is fine and the transfection reagents are brand new(I have tried changing reagents). II plate out cells at 5X10^5 density/well on a six well plate.This gives me 50% confluence on the day of transfection. I found that below this and above this density my transfection efficiency goes down.I was wondering if anyone else has encountered similar problems? Any suggestion will be much appretiated.

#2 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 6,668 posts
565
Excellent

Posted 11 October 2009 - 03:55 PM

I suggest going away from calcium phosphate, unless you have a particular reason for using it, to a lipid micell system such as Fugene or Lipofectamine - much more effective.

#3 wirly

wirly

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 60 posts
1
Neutral

Posted 09 November 2009 - 07:09 PM

Recently I have been having problems carrying out transient transfection of 293T cells using Sigma Calcium Phosphate transfection kits. One day it works and the next day it does not under the exact same conditions. I have tried changing transfection kits, cells, and plasmid DNA. All the effort has failed to shed any light. There will be a day it works and the next it does not. After spending a whole of May trobleshooting this problem I finally got it to work but its stopped working again. I concluded that it must be the state of the cells because the plasmid DNA is fine and the transfection reagents are brand new(I have tried changing reagents). II plate out cells at 5X10^5 density/well on a six well plate.This gives me 50% confluence on the day of transfection. I found that below this and above this density my transfection efficiency goes down.I was wondering if anyone else has encountered similar problems? Any suggestion will be much appretiated.


I agree with bob1, Lipofectamine and the other lipid trasfectants work great, particularly for 293 cells. I'll add that I have found, and it's Invitrogen's dirty little secret, that PEI works just as well as any of their reagents of this class (Lipo-2000, 293fectin, Freestyle MAX etc.), it is 1/100th the price from a chemical dealer and you use the exact same pocedure as the reagents I just mentioned.

#4 cike2008

cike2008

    member

  • Active Members
  • Pip
  • 6 posts
0
Neutral

Posted 03 December 2010 - 09:30 AM

I'm eagar to know how to prepare PEI reagents for transfection.

#5 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 6,668 posts
565
Excellent

Posted 05 December 2010 - 02:24 PM

PEI= polyethylimine.

#6 cellgene

cellgene

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 31 posts
0
Neutral

Posted 19 December 2010 - 10:56 AM

Totally agree, we also use PEI for plasmid transfections, it is easy and works perfect!

But I have never tried it for siRNA transfections and do not know if it works. I still use lipofectamine 2000 for that. Has anyone tried it?


Recently I have been having problems carrying out transient transfection of 293T cells using Sigma Calcium Phosphate transfection kits. One day it works and the next day it does not under the exact same conditions. I have tried changing transfection kits, cells, and plasmid DNA. All the effort has failed to shed any light. There will be a day it works and the next it does not. After spending a whole of May trobleshooting this problem I finally got it to work but its stopped working again. I concluded that it must be the state of the cells because the plasmid DNA is fine and the transfection reagents are brand new(I have tried changing reagents). II plate out cells at 5X10^5 density/well on a six well plate.This gives me 50% confluence on the day of transfection. I found that below this and above this density my transfection efficiency goes down.I was wondering if anyone else has encountered similar problems? Any suggestion will be much appretiated.


I agree with bob1, Lipofectamine and the other lipid trasfectants work great, particularly for 293 cells. I'll add that I have found, and it's Invitrogen's dirty little secret, that PEI works just as well as any of their reagents of this class (Lipo-2000, 293fectin, Freestyle MAX etc.), it is 1/100th the price from a chemical dealer and you use the exact same pocedure as the reagents I just mentioned.






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

©1999-2013 Protocol Online, All rights reserved.