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
- - - - -

What's the longest overhanging primer seq. you have used?


  • Please log in to reply
2 replies to this topic

#1 snpsnooper

snpsnooper

    member

  • Active Members
  • Pip
  • 7 posts
0
Neutral

Posted 18 June 2010 - 09:16 AM

I want to include loxP sites flanking a gene that I want to amplify and then clone it into an expression vector. This is around 34 bp (+ 6 bp for a restriction site) that I must tag on to my primers. Any feedback will be appreciated.

#2 pDNA

pDNA

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 493 posts
14
Good

Posted 18 June 2010 - 11:02 AM

should be absolutely no problem ...use 50bp overhangs without any problems regularly.

Regards,
p

#3 Curtis

Curtis

    Metaller Scientist

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,112 posts
59
Excellent

Posted 19 June 2010 - 09:37 AM

6 mer is quite ok, I've gone up to 8 for overhangs.

A friend of mine used 42 mer primer last time and she had no problem.

in case your PCR product digestion doesn't work, first clone the PCR product into a TA vector, or CloneJet vectors from Fermentas, then digest and clone into the second vector. This way you'll end up with many more colonies.



I want to include loxP sites flanking a gene that I want to amplify and then clone it into an expression vector. This is around 34 bp (+ 6 bp for a restriction site) that I must tag on to my primers. Any feedback will be appreciated.






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

©1999-2013 Protocol Online, All rights reserved.