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 problem

double digest with

  • Please log in to reply
3 replies to this topic

#1 angadbiochem

angadbiochem

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 28 January 2012 - 03:02 AM

Hi
i did a double digest with Ecor1 & BamH1 with a 7Kb plasmid sample prepared through miniprep but not purified.
in 10ul reaction volume i used 2 ul DNA ,1ul10xBSA ,1ulNEbuffer4 and 0.2ul of each enz.
got no expected fragment . these were plasmids taken from transformants of the ligation mix of backbone + fragment.
pls help

Edited by angadbiochem, 28 January 2012 - 04:23 AM.


#2 scolix

scolix

    a student

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 314 posts
5
Neutral

Posted 28 January 2012 - 09:44 PM

Many reasons :
May be the ligation didnot work. Did you mix the enz. tube before pippeting 0.2ul out. The amount of fragment that you wish to see could be too low as the starting material is also too low, 2ul of miniprep DNA.

Better to do a digest in a larger volume, say atleast 20ul.

#3 angadbiochem

angadbiochem

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 29 January 2012 - 08:14 AM

thanks a lot. i did not mix the enzym. yes even i think when i had done 20ul rxn for gel purification i had got a fragment but that was for the original mutant plasmid whose fragment i wish to sub-clone. in 20 ul how much DNA do i take

#4 scolix

scolix

    a student

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 314 posts
5
Neutral

Posted 29 January 2012 - 11:03 AM

You need to take as much DNA so that after the digest the fragment, which is a proportion of the total DNA, is visible on a gel. If the fragment is too small, then you need to have more DNA than if it was big.




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

©1999-2013 Protocol Online, All rights reserved.