QuickHyb (Stratagene): Northern blot problems????
maier_hml at muenster.netsurf.de
Fri Nov 21 09:33:20 EST 1997
tsg1 at york.ac.uk wrote:
>I have used Stratagens'e QuickHyb buffer for northerns for awhile with no
>problems. However, recently
>(since using a new bottle of QuickHyb) i have experienced high backgrounds
>on the northerns. It appears to
>be related to the QuickHyb blocking/hybridization step, since I usually
>mark the position of the RNA makers
>with a pen - and these marks are now clearly visible (white) on a grey
>background. I use BioMax MS (high
>sensitivity film) with o/n exposure and get grey blots. I've used Quickhyb
>with BioMax exposure
>times of 5 days with no problems before (a blot done only a week ago).
>Since I'm looking for a relatively rare
>message- the high background means i cannot detect it!!! Using a probe to
>a highly expressed gene is able
>to detect message - but the background is still visible!!!
-Mix the QuickHyb well before pipetting it on your blot. Perhaps warm
it up to 50°C
- Check the qualitiy of your hering sperm DNA. That was the solution
of the problems we had a time ago.
It is not impossible that you have got a worse bottle of Quickhyb.
Look at the cooled solution, the ratio dextrane sulfate to fluid was
nearly 1:3 when I ´ve used it.
>I was wondering if anyone has experienced this type of problem before and
>if they have any suggestions
>to reduce or overcome them?
>Anyone had experience with other company's rapid hybridization buffers
>and have good/bad comments??
Stratagenes Quickhyb was the best hybridization solution I´ve ever
used for northern blots. In spite of a higher background compared with
classic hybs based on SSC, SSPE or 7% SDS, the signal to noise ratio
was even better than the ratio achieved with Amershams RapidHyb.
>Your comments welcome and thanx for your time.
Hope they were helpfull,
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