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
- - - - -

ligation transformation

ligation transformation

  • Please log in to reply
4 replies to this topic

#1 student47

student47

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 46 posts
3
Neutral

Posted 11 October 2013 - 08:12 AM

hi guys,

you know when you use a enzyme to cut your self ligated vector background by choosing a site in your vector that is not there in your insert. hope you know what am talking about else tell me and i will elaborate.

when i add the cut enzyme, most people say just add a microlitre at end of ligation and incubate at optimum for around 10 minutes, and it should work, is that true? and also

well when you do that, do you need to heat inactivate the enzyme at the end of it? , if i do it might also affect the transformation?  can i just leave it and transform, will it inhibit tranformation?



#2 student47

student47

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 46 posts
3
Neutral

Posted 11 October 2013 - 08:44 AM

thanks in advance..



#3 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,468 posts
247
Excellent

Posted 11 October 2013 - 11:05 AM

That should usually work fine -- most enzymes will work in a ligase buffer. Heat killing will not usually be necessary, but it might help -- some say that heat killing a normal ligation reation improves things, but I've never done that experiment. Do not heat kill ligations with quick ligase buffer (PEG), which will lead to failure.



#4 student47

student47

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 46 posts
3
Neutral

Posted 12 October 2013 - 03:18 AM

thanks phage..



#5 OA17

OA17

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 83 posts
3
Neutral

Posted 12 October 2013 - 04:50 AM

I also think that most enzymes will work in ligase buffer. However, heat inactivation (plus salt removal, of course) is recommended for electroporation. For heat-shock transformation, it is not necessary to inactivate enzymes.







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

©1999-2013 Protocol Online, All rights reserved.