I am using a hybridization buffer for in-situ hybridization which has the following components:
50% formamide, 5X SSC, yeast RNA, heparin and 1% SDS.
Storing the buffer at 4 degrees or -20 degrees causes precipitation due to SDS. The SDS goes into solution upon heating. But, how many times can I heat and use the buffer? Is there any alternative method of storage?
Hi Deborsee,
We make about 300 ml of hybridization buffer and divide it into 30 ml aliquots in 50 ml conical tubes (usually enough for 20 slides).
We store those at -80 and only thaw out enough for each experiment on the day of the experiment.
We thaw/warm the buffer in the hybridization oven (60-75 degrees) before adding it to the slides.
The main concern with using a large stock of buffer is contamination. And since RNAses are everywhere, I would highly discourage from repeated uses of the same stock.
The freeze thaw probably isn't great for the yeast RNA that you are using as a non-specific block; but is less of a problem.
Shanni's technique essentially avoids all problems that you mention. On the other hand, storage of hybridization buffer at minus 20 degrees has no real problems. I normally re-dissolve precipitates in a 37 °C water bath repeatedly without any loss in performance.
Hi, I aliquot my hybridization buffer in 15ml tubes and store them in -20 degrees. As we use 300ul per slide, each tube will be enough for a good 50 slides reaction. As Johannes mentioned earlier storing them in -20 degrees pose no problem at all. I also repeatedly thaw the buffer in 65 degrees water bath prior to use with no loss in performance.
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