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
- - - - -

RT-PCR


  • Please log in to reply
4 replies to this topic

#1 promilasharma

promilasharma

    member

  • Active Members
  • Pip
  • 11 posts
0
Neutral

Posted 17 December 2004 - 09:49 AM

Hi !I am trying to amplify an inducible gene by using RT-PCR kit but either I am not getting the band or if I do slight changes in the conditions I just get the smear towards lower side. though I changed the primrrs but still I am having the problem. Could anyone suggst me something?

#2 littlecell

littlecell

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 56 posts
1
Neutral

Posted 17 December 2004 - 10:08 AM

is your target gene only expressed when it's induced? if it is, i think you'd better find a cell line which constitutively expresses your interested gene. you can check your PRIMER or PCR efficiency with this cDNA. If both work very well, then proceed to your own cell to check its expression profile.

#3 promilasharma

promilasharma

    member

  • Active Members
  • Pip
  • 11 posts
0
Neutral

Posted 17 December 2004 - 10:22 AM

Actually Iam doing in vivo study, not on the cell-line.I just induced the gene in an animal and then after induction isolated RNA from the tissue and then went for RT-PCR.

#4 littlecell

littlecell

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 56 posts
1
Neutral

Posted 17 December 2004 - 11:27 AM

If you don't have the positive control, how can you know the failure of your RT-PCR resulted from the low quality of your RNA, the failure of RT, the failure of induction which means that there's no expression of your interested gene at all, or just PCR reaction? anyway, I think you should find the positive control.

#5 promilasharma

promilasharma

    member

  • Active Members
  • Pip
  • 11 posts
0
Neutral

Posted 18 December 2004 - 03:06 AM

Thanks for your reply.One thing I want to know,is it possible to amplify a target gene of 4.9 kb completely using RT-PCR kit.




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

©1999-2013 Protocol Online, All rights reserved.