Southern blot detection - isotope vs. DIG - what's better - radioactive isotopes or DIG detection? (Mar/15/2006 )

I would say its VERY RISKY to set up a radioactive lab in a room where other things are done. I'm sure everyone working with radioactivity in an academic lab for an extended amount of time has experienced a contamination. And if it's not in a special room, you quickly carry it outside and contaminate people that don't know and therefore will take no measures to decontaminate. Highly irresponsible. Our radioactive lab is very clean, people are well trained but we already had several contaminations: once an epi with radioactive probe popped open while being heated to 95°C and jumped out of the heat block; another time the rubber ring fell out of the lid of a roller bottle before screwing it shut and the radioactive probe + hybridisation liquid leaked into the oven. It's like always with security: you can make things safer but never completely so! And with time the unlikely will happen.




Roche claims similar or superior radioactivity to 32P. I doubt that claim. But I have done both and could see my Southern bands both with DIG+antibody and with 32P. The starter kit is not so expensive. Just give it a go. Maybe you can even convince Roche to give you a test kit, if you say that you are considering changing from radioactivity.

Hope that helps, j

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Roche claims similar or superior radioactivity to 32P. I doubt that claim. But I have done both and could see my Southern bands both with DIG+antibody and with 32P. The starter kit is not so expensive. Just give it a go. Maybe you can even convince Roche to give you a test kit, if you say that you are considering changing from radioactivity.

Hope that helps, j
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thanks for your sharing of experience, j. hope the Roche dealer in my country is as cooperative as yours

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Hi, I already have a thread in this section, but if anyone has a recipe for making P32 hot probes I need it... one that works. My boss wants me to make a radioactive probe by PCR using hot dCTP but I have not been able to get amplification, probably due to the low concentration of the hot dCTP as supplied (3.3 uM stock, when final concentration needs to be 200 uM ). I also have a kit for random primer labeling, but I want to get the PCR method to work first if possible.

Please help

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Hi Wulf,

I would supplement the radioactive dCTP with normal dCTP to get a concentrated dNTP mix for your PCR. This is what they do in random priming kits like High Prime. They don't give you 100% radioactive dCTP there either. This would scorch you.

Many people use the random priming since it's easier; you don't need a PCR machine in the radioactive lab for example. But keep in mind that it probably gives you a Gaussian distribution of probe lengths, say 600-800 if the template is 1000nt.

Hope that helps. All the best,

j

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