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 Digest


  • Please log in to reply
2 replies to this topic

#1 CHurst5841

CHurst5841

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 14 May 2010 - 09:14 AM

Hi,

Does this procedure sound correct for a double digest of plasmid DNA using NotI and Xba1?

3 uL of DNA (2 ug)
4 ul NotI enzyme
4 ul XbaI enzyme
5 ul Promega Buffer D 10X (compatible with both enzymes)
Water to 50 ul

Incubate at 37C for 1 hour.

Thanks!

#2 fishdoc

fishdoc

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 272 posts
2
Neutral

Posted 14 May 2010 - 09:30 AM

Hi,

Does this procedure sound correct for a double digest of plasmid DNA using NotI and Xba1?

3 uL of DNA (2 ug)
4 ul NotI enzyme
4 ul XbaI enzyme
5 ul Promega Buffer D 10X (compatible with both enzymes)
Water to 50 ul

Incubate at 37C for 1 hour.

Thanks!



Never used Promega enzymes, only NEB, but for us, that would be WAY too much enzyme. I rarely use more than 1 ul, even for up to 4-5 ug of DNA.

#3 HomeBrew

HomeBrew

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 930 posts
15
Good

Posted 14 May 2010 - 02:08 PM

I agree with fishdoc -- the total amount of restriction enzyme(s) added shouldn't exceed 10% of the final reaction volume -- I usually try to stay a bit under that. The reason is that most enzymes are shipped stored in a buffer containing 50% glycerol (Promega's NotI, for example, is shipped in 10mM Tris-HCl (pH 7.4), 0.1% Triton X-100, 500mM KCl, 0.1mM EDTA, 1mM DTT, 0.5mg/ml BSA, 50% glycerol, while their XbaI comes in 10mM Tris-HCl (pH 7.4), 300mM NaCl, 0.1mM EDTA, 1mM DTT, 0.5mg/ml BSA, 50% glycerol), thus adding enzyme equivalent to 10% of the final digestion volume makes that digestion 5% with respect to glycerol.

Glycerol concentrations higher than 5% inhibit most enzymes, thus you want to stay below that amount.




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

©1999-2013 Protocol Online, All rights reserved.