Problems PCR-labeling DNA probes with DIG-dUTP
Posted 14 February 2011 - 02:59 PM
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.
Posted 14 February 2011 - 05:57 PM
Posted 15 February 2011 - 09:09 AM
Posted 22 June 2011 - 07:24 AM
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?