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
- - - - -

Digest for insert orientation

Xho1 Sal-HF pGreen

  • Please log in to reply
4 replies to this topic

#1 Tracy0110

Tracy0110

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 22 July 2012 - 06:01 PM

Hi,

I digetsed my plasmid (pGreen) and my inserts with the Xho and Sal enzymes and ligated them together before transforming e.coli. The insert appears to be present in the plasmid however I now need to check the orientation of the insert as Xho and Sal have the same 'sticky ends' and my cloning is therefore not directional.

Is it possible to check the orientation of my insert using a restriction enzyme digestion with these enzymes? Or should I just sequence it?

#2 Tracy0110

Tracy0110

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 22 July 2012 - 07:29 PM

I think I've worked this one out for myself. By digesting with either Xho or Sal and digesting with an enzyme which cuts within my insert, I can work out which way it has gone in depending on the sizes I get from the digestion

#3 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,709 posts
398
Excellent

Posted 22 July 2012 - 07:35 PM

Correct. You should still sequence the insert if you PCR generated it, just to make sure there were no errors generated.

#4 Kamran

Kamran

    Kamran

  • Active Members
  • PipPipPipPipPip
  • 41 posts
2
Neutral

Posted 22 July 2012 - 09:50 PM

you can also perform colony PCR using vector-insert primers to confirm correct orientation.

#5 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,466 posts
247
Excellent

Posted 23 July 2012 - 02:50 AM

One of the directions of insertion will not cut with either enzyme. The other orientation will cut with both.





Also tagged with one or more of these keywords: Xho1, Sal-HF, pGreen

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

©1999-2013 Protocol Online, All rights reserved.