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
- - - - -

Problems with the preparation of radiolabelled Probe


  • Please log in to reply
2 replies to this topic

#1 shivasankari

shivasankari

    member

  • Active Members
  • Pip
  • 11 posts
0
Neutral

Posted 18 November 2010 - 05:44 AM

Hi I am having a problem in the preparation of radioactive DNA probe.I have been trying to prepare DNA fork - 32 P labelled, for the last few week. The problem is I always get single strand contaminants with the double strand probe. I am not able to eliminate this. This is the protocol I follow :
1) 20U single strands (5 micromolar) is labelled with 32 P using T4 polynucleotide kinase, for 45 minutes. Stop the reaction with EDTA.
2) I anneal 2 fold excess of 20D strand (5 micromolar) with the labelled 20U, keep it in 95 deg for 10 mins and then leave it to anneal overnight at room temp.
3) Purify the probe and load aliquots before and after purification.

I have tried changing the annealing buffer, fresh dilution of the single strands, and stuff like that , but still I get the single strand contaminants in the gel.Since I will have to do a helicase assay with this probe, I will definitely need to get rid of this single strand contaminants. If someone could help me it would be great!! I am desperate to make my expt work!!

thanks in advance
Reg, Shiva
Cheers

Shiva

#2 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,623 posts
388
Excellent

Posted 21 November 2010 - 04:29 PM

Aren't there DNAses that are single strand specific?

#3 shivasankari

shivasankari

    member

  • Active Members
  • Pip
  • 11 posts
0
Neutral

Posted 22 November 2010 - 05:27 AM

[quote name='bob1' timestamp='1290385769' post='92838']
Aren't there DNAses that are single strand specific?
[uote]
Hi Bob,

Thanks for the reply. I am not sure them. But the problem in using them is that, when I am using them in my helicase assay, which is supposed to give me the degree of unwinding, I am scared that my Single strand products will also be cleaved by the nucleases! So, I guess I might not be able to use them.

Thanks

Cheers

Shiva
Cheers

Shiva




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

©1999-2013 Protocol Online, All rights reserved.