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
- - - - -

in silico primersecondary structure issues.

qPCR; Primer dimer

  • Please log in to reply
No replies to this topic

#1 tsalihah

tsalihah

    member

  • Members
  • Pip
  • 1 posts
1
Neutral

Posted 02 April 2014 - 04:39 PM

Hi,

 

 I am very new at designing primers and qPCR and I need all the help I can get.

 

I am designing primers using idtdna primerquest (for qPCR and SYBR Green) and run the subsequent primer pairs on the oligoanalyzer for secondary structure.

 

I use the following rules to rule out primer pair (for presence of secondary structure in silico):

 

hairpin:

1.3’end pairing : ∆G≥  -2 kcal/mol

     Or

     Internal pairing : ∆G≥ -3kcal/mol

 

dimers (self/hetero):

1.3’end pairing : ∆G≥ -5 kcal/mol

     Or 

     Internal pairing : ∆G≥ -6 kcal/mol

 

and for hairpin and dimers, Tm of secondary structure  atleast 5oC≤ Ta primer

 

but the end result left me with no primers at all. I know that anything ∆G≥-9 kcal/mol is also acceptable, could I make exception for dimers that had -6 to -9 kca/mol? or should I just cut my loses design a whole new batch of primers?






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

©1999-2013 Protocol Online, All rights reserved.