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!
- - - - -

primers with tags how to decide wich nucleotides

  • Please log in to reply
No replies to this topic

#1 gala84



  • Members
  • Pip
  • 2 posts

Posted 12 August 2011 - 03:55 AM

Hi to everybody, I'm new in this forum and I'm also new in molecular biology.
I work on marine bacteria involved in Nitrogen Cycle and from environmental samples I have to do PCR on specific functional genes. I have to work both with DNA than cDNA and on these selected amplicons do pyrosequencing 454. To distinguish the samples from DNA and cDNA I should add a tag of 5 nucleotids for each primer (forward and reverse) that needs to be different for DNA and cDNA. So when I'll have my sequences I know from which samples they come.
Probably i didn't explain it very well.... sorry!!!!

My questions are:

1. With these tags, since the lenght is higher, is it necessary to modified the annealing temeperature???
2. The tags mustn't do dimers and secondary structures with the sequence. How I have to choose the correct nucleotides to put at the end of each primers? The company where I order them can do this? Or I have to do by myself? and How?

thank you in advance


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

©1999-2013 Protocol Online, All rights reserved.