Problems with the preparation of radiolabelled Probe - Hi, (Nov/18/2010 )

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

Aren't there DNAses that are single strand specific?

bob1 on Mon Nov 22 00:29:29 2010 said:

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