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 A260/230 problem


  • Please log in to reply
2 replies to this topic

#1 joy123

joy123

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 105 posts
3
Neutral

Posted 27 August 2012 - 06:16 AM

Hi! I extract genomic DNA (both human and bacteria, and I am interested in bacteria) from human liquid specimen, and plan to do qPCR with it.

The general DNA extraction procedure is: lysozyme-->protease K-->cell lysis-->protein precipitation-->centrifugation-->Isopropanol DNA extraction--> rinse with 70% Ethanol-->dry-->resolve with 10mM Tris (pH8.0).

The result turns that, the A260/280 is ~1.8. But the A260/230 is very low, ~0.9-1.4. The peak at 220 is high (attached).

I tried DNA precipitation with Isopronanol to get rid of salt, and resuspend in Tris, but the A260/230 is still low.

Then, I use sodium acetate+Ethanol to get DNA precipitaiton, and resuspend in Tris, but the A260/230 is still low.

My questions are:
1. Will it interfere with qPCR efficiency? Do I need to resolve the low A260/230?
2. Would you please give me some suggestions how to resolve this problem?

Thanks a lot!

Attached Thumbnails

  • picture.jpg

Edited by joy123, 27 August 2012 - 06:25 AM.


#2 AquaPlasmid

AquaPlasmid

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 74 posts
7
Neutral

Posted 27 August 2012 - 04:58 PM

The scans look normal to me. What were the DNA concentrations? Try use 10x higher concentrations.

#3 joy123

joy123

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 105 posts
3
Neutral

Posted 28 August 2012 - 08:39 AM

The concentration is ~200ng/ul. I don't know what may caused the high peak at 220nm. And I wonder if it will interfere with qPCR assay.

The scans look normal to me. What were the DNA concentrations? Try use 10x higher concentrations.






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

©1999-2013 Protocol Online, All rights reserved.