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
- - - - -

double digestion with sac1, hindIII


  • Please log in to reply
3 replies to this topic

#1 mbsea

mbsea

    member

  • Active Members
  • Pip
  • 24 posts
0
Neutral

Posted 14 October 2011 - 03:52 AM

Hi everybody, i have been struggling with my ligation of my gene into oet 21a vector. Now i changed my primers also enzymes. I want to use sac1 and hind III fermentas enzymes. I cloned my gene into pcr 8 topota vector. Did anybody use this combination and offer me a protocol that how long must i wait and how much enzyme must i use?

best regards.

#2 Papaver

Papaver

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 77 posts
19
Good

Posted 14 October 2011 - 05:35 AM

Fermentas double digest: http://www.fermentas...ls/doubledigest
Select your enzymes, then fermentas show their recommendation and general properties of the enzymes.

I usually digest ~ 1 µg of template, 1xbuffer, 0.5 - 1 µl enzyme in a volume of 30 - 50 µl. Usually digestion at 37 °C for 2-3 h is enough.

Edited by Papaver, 14 October 2011 - 05:36 AM.


#3 mbsea

mbsea

    member

  • Active Members
  • Pip
  • 24 posts
0
Neutral

Posted 16 October 2011 - 11:30 PM

thank you very much. I was wondering about the incubation time because i am not sure that my vector is digested well..
best regards...

#4 Papaver

Papaver

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 77 posts
19
Good

Posted 16 October 2011 - 11:37 PM

If you are not sure how long would be ideal you can set up several digestions and stop the reaction after 1, 2, 3...hours. I made the experience that too long incubation and/or too much enzyme ends up in unspecific digestion of my plasmid. Too less can lead to uncomplete digestions which I prefere at the end as long as enough of my plasmid was digested properly. It's always learning by doing...




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

©1999-2013 Protocol Online, All rights reserved.