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
- - - - -

Why would you use DNase as a control in dot blot assay confirming viroid import?


  • Please log in to reply
1 reply to this topic

#1 sara_molecular

sara_molecular

    member

  • Members
  • Pip
  • 1 posts
0
Neutral

Posted 27 June 2014 - 12:32 PM

So I'm working on the CChMVd. After producing an RNA copy by transcription I then labelled by RNA with biotin. I grew pea plants, and performed a maxi prep for chloroplasts. I induced an import reaction between the labelled RNA and chloroplasts. I purified the solution and used Trizol to remove DNA. I then separated the chloroplast using centrifugation. I applied RNAse as a control to the chloroplast pellet resuspended in TE buffer, and dh2o in another sample. I understand this is to establish how much of the RNA was imported in the chlorplast against how much was not imported in the chloroplasts. To the supernatant of the chloroplast pellet I used DNase in one sample, and again dh2o in the other. I was advised to do this as a control but I am unsure of the reasoning. Why would there be labelled DNA in the supernatant? Why do I need to use DNase as a control? What does the signal from a dot blot assay for the RNA signal in the supernatant tell me vs. the signal with supernatant treated with DNase? Any ideas?



#2 sara_molecular

sara_molecular

    member

  • Members
  • Pip
  • 1 posts
0
Neutral

Posted 27 June 2014 - 12:32 PM

So I'm working on the CChMVd. After producing an RNA copy by transcription I then labelled by RNA with biotin. I grew pea plants, and performed a maxi prep for chloroplasts. I induced an import reaction between the labelled RNA and chloroplasts. I purified the solution and used Trizol to remove DNA. I then separated the chloroplast using centrifugation. I applied RNAse as a control to the chloroplast pellet resuspended in TE buffer, and dh2o in another sample. I understand this is to establish how much of the RNA was imported in the chlorplast against how much was not imported in the chloroplasts. To the supernatant of the chloroplast pellet I used DNase in one sample, and again dh2o in the other. I was advised to do this as a control but I am unsure of the reasoning. Why would there be labelled DNA in the supernatant? Why do I need to use DNase as a control? What does the signal from a dot blot assay for the RNA signal in the supernatant tell me vs. the signal with supernatant treated with DNase? Any ideas?






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

©1999-2013 Protocol Online, All rights reserved.