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
- - - - -

Amplification of 3.4 kb product from RNA for cloning


  • Please log in to reply
5 replies to this topic

#1 oms

oms

    member

  • Active Members
  • Pip
  • 9 posts
0
Neutral

Posted 26 December 2010 - 05:02 AM

I am trying to amplify the complete coding region of a particular gene of 3.4 kb size. I want to PCR clone it into an IRES2 -gfp cloning vector. To the primers again 15bp homologous sequences have been added. The Tm of bothe the primers minus the 15bp homologous bases is around 56 deg. GC content is 65%. I started with 5ug/ul RNA and used Titan One step PCR Kit from ROCHE which contains AMV RT as well as Hi-Fi Polymerase and can amplify upto 6kb long product. But still Im not getting any bands. Can any one help me?

#2 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,373 posts
226
Excellent

Posted 26 December 2010 - 06:58 AM

It's the Tm of the region matching the template (homologous region) that matters. This likely needs to be longer than 15 bp for normal PCR conditions. Did you really mean that 15 bp were added to the 5' end of a primer otherwise matching a template?

#3 oms

oms

    member

  • Active Members
  • Pip
  • 9 posts
0
Neutral

Posted 26 December 2010 - 11:35 PM

I used Clontech infusion primer design software where infusion primer design tool converts the existing primers (which in my case was 20 mers long with above mentioned GC content and Tm)into infusion primers by adding vector specific sequences (15mers long)on the 5'side of the primers.So then the modified primer becomes 35bp long.
So what Tm shd I consider? Inclusive of this extra bases (which the clontech protocol says u dont have to). I really dont know. I am not able to amplify my gene of interest using these primers.

#4 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,373 posts
226
Excellent

Posted 27 December 2010 - 07:53 AM

Your initial pcr cycles will only bind the primer with the 20 bp of template matching primer DNA. That's the region that will control your initial Tm. But usually people worry FAR too much about Tm. 55C works most of the time for pcr annealing. If it doesn't, then there is often something else wrong.

#5 oms

oms

    member

  • Active Members
  • Pip
  • 9 posts
0
Neutral

Posted 27 December 2010 - 09:57 PM

Should I try taking large amount of template (5-10ug/ul) as a starting template, will that help?

#6 ElHo

ElHo

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 90 posts
1
Neutral

Posted 05 January 2011 - 05:16 AM

Did you analyze the expression of your gene of interest in the sample you are using (for example by realtime PCR)? Is your gene of interest expressed in sufficient amounts? Sometimes reamplification of your pcr product using the same primers helps to get a product if expression is low. But you may encounter mutations in your insert due to misincorporations (even a proofreading polymerase is not 100% perfect)
Doing two step RT-PCR I also often had problems to amplify larger fragments (>1 kB) from cDNA. In my opinion the cDNA one gets after reverse transcription is kind of fragmented, making amplification of longer products difficult.




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

©1999-2013 Protocol Online, All rights reserved.