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
- - - - -

Why perform restriction enzyme digestion again?

restriction enzyme

  • Please log in to reply
1 reply to this topic

#1 Benny85

Benny85

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 16 March 2012 - 07:11 AM

I have bacteria stocks/clones that contain my plasmid of interest. The clones were previously tested to contain the plasmid of interest by restriction enzyme digestion.

Now that i need to use the plasmids again. So I inoculated the bacteria stocks onto my ampicilin bacteria media (bacteria carry this plasmid of interest with ampicilin resistance gene) and let the bacteria propagate, then did a maxi prep to obtain the plasmid.

Why is there a need to perform restriction enzyme (RE) digestion again on the plasmid obtained (which has already been tested)?

Is there something to do with the bacteria losing the plasmid during propagation? how? are there any other reasons to perform RE again?

Thanks.

Benny85

#2 Trof

Trof

    Brain on a stick

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,199 posts
109
Excellent

Posted 16 March 2012 - 08:35 AM

Bacteria can do all sort's of things with a plasmid. They can loose it, they can recombine it. Also you used a different prep, isolation, different conditions. I would check it.

Edited by Trof, 16 March 2012 - 08:35 AM.

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon






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

©1999-2013 Protocol Online, All rights reserved.