Re-using hybridisation solutions containing double-stranded probes?
jlitts at onyx-pharm.com
Mon Mar 6 11:59:03 EST 1995
In article <3j6587$3fg at quandong.itd.adelaide.edu.au>,
magi at smug.student.adelaide.edu.au (Void) wrote:
> DREWES at MPASMB.DESY.DE wrote:
> : Has anybody ever tried re-using plaque hybridisation solutions containing
> : 32P-labeled double stranded probes? It would mean a significant reduce in
> : radioactive waste and expensive stuff like salmon sperm DNA and deionized
> : formamide. I am thinking about simply storing the used solution in the freezer
> : and then denature the DNA and the probe again by boiling for ten minutes before
> : the next use.
> : Thanks for any suggestions...
Yes, I have done this numerous times with good success. You can use the
probes for a couple weeks or more. Also, you might note that if the sample
is in Formamide hybridization solutions it is not necessary to heat the
solution to boiling to melt the dsDNA. This is particularly useful in
experiments where multiple probes are pooled to go through a library. Make
the several probes, reserve about 10% of each for the final screen, and
pool the remainder at about 1E6 cpm/ml of each probe. Purify all the
plaques with the mixed probe through two or three rounds, and then once you
have purified plaques, plate them out in a grid which you lift replicates
according to the number of probes in the pool. Each replicate lift is then
probed with the 10% reserved probe, and voila, multiple isolated clones.
Good luck and happy hunting.
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