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!
- - - - -

Nco1 digest problem

  • Please log in to reply
4 replies to this topic

#1 spellberg56



  • Active Members
  • Pip
  • 13 posts

Posted 25 March 2010 - 12:38 PM

Hello, I am screening colonies for an insert by digestion with Nco1. My plasmid preps have yield ~.3 ug/ul. For each reaction I have: 4 ul plasmid, 1ul #3 buffer, .2 ul Nco1, 4.8 ul H20 for a total of 10ul. I digest for ~2 hours at 37 C. When I run my samples on the gel I just get a smear of DNA in the 100 bp to 500 bp range. I do expect a band at 300 bp but also a 1kb band and 4 kb band. I also have checked my plasmid on a gel and analyzed DNA content spectroscopically. Why am I getting this smear?

Thank you.

#2 swanny



  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 368 posts

Posted 25 March 2010 - 02:49 PM

First up, I'd try a different lot of NcoI. If you get the same problem, try a different enzyme. If that still gives a smear, try reprecipitating the DNA. I just hope you have enough DNA for all of these tests...
Heart disease kills more women than breast cancer, but heart attack symptoms differ from men's symptoms. Get to know your heart... it could save your life.

#3 tea-test



  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 169 posts

Posted 26 March 2010 - 07:05 AM


are you using 0.2Ál enzyme? then maybe try to use less DNA like 250ng. If you are using 2Ál increase the rxn volume to at least 20Ál.
tea-test: The artist formerly known as Ned Land

#4 Qundo12


    E. coli farmer

  • Active Members
  • PipPipPipPipPip
  • 88 posts

Posted 27 March 2010 - 12:43 AM

I think you had a very high enzyme:DNA ratio. This could lead to the star activity. To overcome that, you can add the total volume to 20ul, but since the DNA amount is very low, you need to increase the DNA also. I suggest to use 10ul plasmid DNA, 2ul buffer, .1ul NcoI, 7.9ul H2O and incubate less than 2h. Also, another problem you may get is the endogenous enzymes of the host (endA+ strain) if you used the wild type strain, in this case, you should add one buffer in the midipreps kit to exclude the enzymes.

#5 phage434



  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,747 posts

Posted 27 March 2010 - 07:48 AM

The fraction of your digestion reaction that is composed of your DNA solution is very high. Any contaminants in your DNA can easily inhibit the reaction. I would do this reaction in 50 ul volume, with 4 ul DNA, 1 ul RE, 5 ul buffer, 40 ul water. In this reaction, 80% of the volume is water, with no impurities. The lower concentration of enzyme (and hence also lower concentration of glycerol) also helps. This is what your enzyme manufacturer recommends, also, I might add. I would probably do this with NcoI-HF, and switching to buffer 4.

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

©1999-2013 Protocol Online, All rights reserved.