Southern blot detection - isotope vs. DIG - what's better - radioactive isotopes or DIG detection? (Mar/15/2006 )
I am wondering which DNA blot (Southern blot) detection method is most sensitive & most specific.
Did anybody compare radioactive DNA probes with DIG chemiluminescent detection? Roche provides a kit for the latter & states: "0.03 pg of homologous DNA is detectable with chemiluminescence (probe concentrations at 20 ng/ml). A single copy human gene (t-PA) is detectable in a Southern blot, loading 1 µg of digested placenta DNA."
What's your experience? Do you prefer isotopes or non-radioactive labeling to detect elusive DNA fragments? Let me know.
I prefer isotopes.
At my old department we tried DIG, but it didn't seem to work as well as radioactivity. There were always some people who got DIG to work and with others it never worked. We still don't know why.
With radioactivity you always get signal (or you must have forgotten something). And with the scanners you can "optimize" the signal (longer/shorter exposure and darker/lighter settings). I don't know how that is with DIG.
But of course there are also disadvantages for radioactivity. Just being radioactive itselve. You have to have a special lab and diploma's for it and be careful. I would go for the DIG if there is not a special labspace for isotopes.
Thanks for the relpy. That's what a postdoc here said as well. But then I had trouble even with the radioactive blot. If your cells are not already waiting to be torn opened and have their genomic DNA digested and blotted, there's also a timing problem with decaying radioactivity.
You can optimise DIG signal if you are using a chemiluminescent method similar to the phosphoimager for isotopes. That is not possible for DIG colour reactions though.
So, you guys didn't find out why some DIG detections didn't work?
I like a chemi method involving biotinylated oligos, I use NEB's phototope star
I have never pushed the limits of detection though...may not be as sensitive as radioactivity?
Definitely DIG for me, 2 hr exposure as opposed to overnight for isotope, as well as the convenience of not having to deal with radioactives and being able to label by any method I like, including PCR.
thanks for the reply. Classic biotin - streptavidin - alkaline phosphatase.
If anybody wants to read more about it follow this link to the NEB website:
They say there exposure time 1-10min & you can re-expose if you need to.
Did you ever compare it to DIG or radioactivity?
thanks a lot for replying. Great to hear that DIG method works for you. You are using stuff from Roche?
If anybody wants to read more about DIG, Roche has a nice website with intro & advanced info:
Did you ever compare?
Roche shows a comparison of DIG & 32P on their website:
I think this comparison is problematic since almost no details are given for the radioactive detection, plus the membranes used are hard to compare - more lanes & lower magnification of the radioactive blot, for example.
P32 labelling is more sensitive, for sure.
This is what I've heard from all the labs that run Southern Blots frequently: they see no point in using DIG probes.
lucky for me to chance upon this discussion on radioactive vs nonradioactive labeling
i don't have any experience in radioactive work but my new lab boss is very enthusiastic abt it and setting up a room/corner for it. which freaks out the rest of the lab!
can't nonradioactive labels work as sensitively or consistently as isotopes? for southern and northern hybs.
may i know if a special room with protective walls etc is crucial/imperative for prevention of the exposure? anyone who can suggest how to set up a place for doing it earns my biggest gratitude
err, just for curiosity sake: any lab out there which's working with radioactive detection "unprotected"?!