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
- - - - -

Reverse Sequences


  • Please log in to reply
2 replies to this topic

#1 Mulletman

Mulletman

    member

  • Active Members
  • Pip
  • 9 posts
0
Neutral

Posted 24 September 2009 - 01:36 AM

Hi all
a questions regarding the orientation of sequences during cloning - a few months ago i cloned some PCR fragments (Topo10, thermal method), and the sequences came back as expected (aligned perfectly with the reference sequences).
A few weeks ago a new set of clones (different PCR product, Topo10, electroporation method) were sequenced, and nearly half of the sequences needed to be reversed for alignment (after which they aligned perfectly).

What causes selection of strand orientation during the cloning process? Should i try to improve PCR eficiency, ligation or what?
Has anyone else had similar experiences?

#2 Jackson

Jackson

    member

  • Members
  • Pip
  • 1 posts
0
Neutral

Posted 24 September 2009 - 03:59 AM

Hi all
a questions regarding the orientation of sequences during cloning - a few months ago i cloned some PCR fragments (Topo10, thermal method), and the sequences came back as expected (aligned perfectly with the reference sequences).
A few weeks ago a new set of clones (different PCR product, Topo10, electroporation method) were sequenced, and nearly half of the sequences needed to be reversed for alignment (after which they aligned perfectly).

What causes selection of strand orientation during the cloning process? Should i try to improve PCR eficiency, ligation or what?
Has anyone else had similar experiences?

When you clone a PCR fragment the orientation in the clone is random. Just look for your primers in the sequence. If your forward primer is still in the same orientation than you don't need to complent inverse your sequence.

Regards,
Jackson

#3 Chimp

Chimp

    member

  • Active Members
  • Pip
  • 14 posts
0
Neutral

Posted 25 September 2009 - 02:33 AM

Yes, direction of insertion into TOPO is completely random, no RE sites used....




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

©1999-2013 Protocol Online, All rights reserved.