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
- - - - -

Purification of DNA after MeDIP and WGA


  • Please log in to reply
1 reply to this topic

#1 Abovethegenome

Abovethegenome

    member

  • Members
  • Pip
  • 1 posts
0
Neutral

Posted 18 July 2011 - 10:34 AM

Hi,
I'm currently attempting to run some DNA methylation arrays. So far I extracted my DNA using TRIzol (followed by cleanups), since I needed to get both DNA and RNA from the same tissue. I then performed MeDIP using a kit from Epigentek and confirmed a successful IP by running a PCR for H19 and B-actin. After that I amplified my DNA using Sigma's WGA2 kit and purified the product using Qiagen's qiaquick PCR purification kit. Most of my samples came out with high concentrations (~70ng/ul in 50 ul) and with good 260/280 ratios (~1.8). However, my 260/230 ratios are very low (~0.7). I've tried contacting all the companies but so far none of their solutions have worked. Qiagen recommended running the samples through the kit again, but this time making sure they are at room temp and performing the wash twice, however this yielded the same result. There is also a spin to remove any leftover alcohol, so I am not sure about what is contaminating my samples. I also confirmed my readings using another nanodrop. Has anyone here had a similar experience and/or have any advice?

#2 Muhammad Umer

Muhammad Umer

    member

  • Active Members
  • Pip
  • 29 posts
0
Neutral

Posted 11 September 2011 - 12:15 PM

Hi
I am not sure if my answer would help you. i have faced similar issue of awkward 260/230 ratios after purification with same qiaquick kit (for some other experiment). I was told by a colleague that it happens sometimes and it is not a big issue. There is some unknown component in Qiagen buffers which sometimes causes this problem. This should not worry you I think because I carried out pyrosequencing platform/reaction based experiment on such samples and they worked quite fine, at least similar to those which had normal 260/230 ratios




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

©1999-2013 Protocol Online, All rights reserved.