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
- - - - -

Pair of primers (SYBR) with more than one amplicon product(?)


  • Please log in to reply
4 replies to this topic

#1 thinker

thinker

    member

  • Active Members
  • Pip
  • 16 posts
3
Neutral

Posted 27 March 2013 - 04:11 AM

Hi everyone,

I have a question.

I am trying to design a pair of primers in a repetitive sequence, but i am facing the issue below:

The forward primer is ok and align in only one site in the sequence.

But the reverse primer align in four sites downstream the sequence amplifying 4 amplicons: 213bp, 425bp, 637bp and 849bp.

My question is: at the end of PCR, how many amplicons will I have?

Theoretically I know that I will have 4 amplicons, but I was thinking if the first aligment of the reserve primer (213bp) will allow the other bigger amplicons to be produced.

What do you think about it?

Thanks,

Thinker

#2 bigudukaz

bigudukaz

    member

  • Active Members
  • Pip
  • 27 posts
2
Neutral

Posted 27 March 2013 - 04:15 AM

does the second primer aneal to the sequence that is 100% complementary (in all 4 cases) ?

#3 thinker

thinker

    member

  • Active Members
  • Pip
  • 16 posts
3
Neutral

Posted 27 March 2013 - 06:20 AM

does the second primer aneal to the sequence that is 100% complementary (in all 4 cases) ?


Yes bigudukaz
It is a repetitive sequence, so the primer align perfectly in all 4 downstream sequence.

#4 bigudukaz

bigudukaz

    member

  • Active Members
  • Pip
  • 27 posts
2
Neutral

Posted 27 March 2013 - 06:54 AM

You will amplify all of the 4 products and the melting curve will look funny. Probably the only option would be to use probe for the shortest fragment - you will get around the problems because of 4 amplicons forming and you still should be able to quantify gene expression.

There will be the most of shorter fragments and least the longest ones.

I haven't tried such experiment yet, but time will come.

#5 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,469 posts
247
Excellent

Posted 28 March 2013 - 03:22 AM

You may be able to select the shorter sequence by using a shorter extension time. It will be difficult to selecct the longer sequence. The PCR reaction will favor the short sequence, and you may find it difficult to produce much of the longer sequences, but will likely get some of the longer fragment.




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

©1999-2013 Protocol Online, All rights reserved.