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Best Southern Blot detection method???

Southern Blot

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#1 ESH

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Posted 18 July 2012 - 11:24 AM

Hi,

I will be doing a Southern Blot and would like to know what is the best detection method for Southerns. My head is swimming a bit but from what I understand the most common are radioactive probes, flourescein, DIG, biotin, alkaline phosphatase, etc. What are the main differences between each one and which final detection method (ie chemiluminescence, direct flourescence labeling/flourescein visualization, "indirect" flourescence labeling [ie using anti-flourescein etc.], and radiation detection) is the most sensitive? Any help would be appreciated!

#2 bob1

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Posted 18 July 2012 - 01:49 PM

Radiation is probably still the most sensitive due to the long time you can put exposures down for (weeks even), though chemiluminescence (a few hours max) and DIG labelling are pretty close. Radioactive labelling comes with the hazard of playing with radioactive materials and the subsequent disposal and health risks.

I personally like DIG labelling with a chemiluminescent secondary detection probe, as it is very easy to generate PCR based DNA probes which are DIG labelled extensively (rather than the single label you get with terminal transferases), making it quite easy to detect the signal and not much trouble, unlike working with radioactive probes.





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