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
- - - - -

low DNA yield after DNA digestion and gel extraction

Digestion Dna yield gel extraction

  • Please log in to reply
No replies to this topic

#1 KhaleesiDany

KhaleesiDany

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 33 posts
1
Neutral

Posted 23 January 2015 - 01:19 AM

greetings 

 

i digested a plasmid to extract my sequence of interest however i always get a faint band which means low dna. I tried increasing amount of dna digested to 2 ug but the band of interest is still faint but the top band/back bone is very bright and big.. i suspect there wasn't enough digestion? i used a 30 uL reaction volume for 2 ug DNA and 2 hour  incubation period and NEB enzyme where am i going wrong? i need  a lot of insert DNA

 

Another prob when i digest my vector with the backbone my yields aren't to great either but they sufficient and then i do dephosphorylation and after column clean up i have lost 50% of my DNA how do i reduce the amount of lean up steps because the process was: digest, gel extraction that included column purification, and then dephosphorylation followed by another column purification step.... in the past i did dephosphorylation directly after digestion because it was a thermo scientific product that would work in thermo scientific enzyme buffer but now we use NEB enzyme and i dint think thermo dephosphorylation kit works in NEB buffer. 


Edited by KhaleesiDany, 23 January 2015 - 01:29 AM.






Also tagged with one or more of these keywords: Digestion, Dna yield, gel extraction

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

©1999-2013 Protocol Online, All rights reserved.