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
- - - - -

Finding the right sequence

BLAST gene sequence PCR

  • Please log in to reply
4 replies to this topic

#1 Rachel Ruhlen

Rachel Ruhlen

    member

  • Active Members
  • Pip
  • 3 posts
0
Neutral

Posted 16 February 2012 - 08:15 AM

I've been having trouble with RT-PCR and have finally ruled out everything except the primers. I ordered control RNA and control primers and got the expected band there, and I'm fairly confident I have good RNA (nice bright 28S and 18S bands), so I'm pretty sure the problem is my primers.

My goal is to confirm the results of the genome microarray experiments that were done by someone else. This is for rat spinal cord RNA expression.

To start I selected an internal standard, based on the literature I picked a ribosomal RNA, Rpl27. I used genbank to identify the sequence and came up with

NM_022514. I put that into PrimerQuest (primer design tool available on Integrated DNA Technologies website). That gave me 5 primer sets and I ordered all 5 and none of them gave me a band.






Any suggestions?

#2 Trof

Trof

    Brain on a stick

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,205 posts
110
Excellent

Posted 16 February 2012 - 08:40 AM

Well none of them gave you a band... did you try to optimise the reaction? What annealing temperature you used?
The gene is not GC rich, and the primers look fine. Ta 60 should work.

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#3 Rachel Ruhlen

Rachel Ruhlen

    member

  • Active Members
  • Pip
  • 3 posts
0
Neutral

Posted 16 February 2012 - 08:46 AM

I used 60C for the annealing temperature.
I tried adding a ul of Mg to the buffer.

#4 ickypimp

ickypimp

    member

  • Active Members
  • Pip
  • 14 posts
1
Neutral

Posted 16 February 2012 - 03:51 PM

so titrate in the mag chloride

#5 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,506 posts
253
Excellent

Posted 16 February 2012 - 06:56 PM

I strongly suggest trying a 55C annealing temperature. It mostly works.





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

©1999-2013 Protocol Online, All rights reserved.