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Problems PCR-labeling DNA probes with DIG-dUTP


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#1 labstarloz

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Posted 14 February 2011 - 02:59 PM

Has anyone out there had success using Roche's PCR DIG Probe Synthesis Kit for labeling PCR products?

I have 6 different DNA probes I'm trying to label by PCR for use in Southern blotting. All are < 500 bp in length, 30-50% GC content. I spent considerable time optimizing the PCR conditions for each template/primer set using the Roche kit's enzyme and reagents, and can reliably amplify each probe using standard dNTP mix. However, under the same PCR conditions, I cannot get any of the primer sets to generate a DIG-labeled product when I add back the DIG-dUTP labeling mix (as determined by running labeled and unlabeled PCR reactions side-by-side on a gel). I can amplify the included control PCR product without any problem, and all of the unlabeled PCR reactions amplify as expected. I just can't seem to get any product when I incorporate the labeling dNTP mix.

Is there something I'm missing in the protocol? I'm post-staining my agarose gel with EtBr (0.5ug/mL), but assume that the labeled product should amplify almost (if not equally) as well as the unlabeled product, and should therefore be similarly detectable? I checked the troubleshooting notes - Roche suggests diluting the dNTP labeling mix for PCR products >1kb in length - has anyone tried this and had success labeling smaller length probes?

Any suggestions or feedback would be welcome.

Cheers,

#2 phage434

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Posted 14 February 2011 - 05:57 PM

What enzyme are you using? Make sure it is either Taq or a Taq + Pfu or similar mix. dUTP will not be incorporated well by pure Pfu or Phusion or other proof reading enzymes. I always succeeded just spiking a small amount of dig-dUTP into a normal pcr reaction with Invitrogen PCR supermix high fidelity master mix (a mix of Taq and Pfu).

#3 labstarloz

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Posted 15 February 2011 - 09:09 AM

I'm using the Expand High Fidelity enzyme mix provided with the kit, which is a mix of Taq/Pwo I believe.

#4 RebeccaW

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Posted 22 June 2011 - 07:24 AM

I was interested to read your findings as I have had considerable difficulty DIG labelling probes recently. We have been using this kit for several years and it always worked perfectly.

Several months ago (around feb 2011) we had to reoptimise the labelling reaction, as using our previous conditions we did not get a labelled product. I managed to get a labelled product using 1/4 the amount of DIG in the reaction than used previously.

I would be interested to know if you managed to get a labelled product?

Have you contacted the company?




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