Frank O. Fackelmayer
Frank.Fackelmayer at uni-konstanz.de
Mon Feb 14 08:55:02 EST 2000
> Hi! I'm planning to use a spot blot for RNA with a DNA probe (labeled
> with BrightStar psoralen-biotin kit from Ambion) . My professor insists
> that we make the hybridization, wash, and blocking buffers instead of
> using their BioDetect BrightStar kit.
good idea to save lots of money!
> I'm concerned that the procedure
> she wishes to follow (Maniatis) may not be compatible with this setup
> because it is intended for radioactive probes. In particular, the
> blocking reagent requires sheared salmon DNA and BSA. I found another
> protocol in Ausubel specifically for nonradioactive probes on a neutral
> membrane. However, the blocking buffer was quite different, requiring a
> high concentration of SDS (5%), which I am concerned may distrupt the
> positively charged membrane. Furthermore, this buffer did not contain
> protein or DNA.
> Should one of these procedure work
Yes, both will work well. You don´t have to be afraid of destroying the
membrane by high SDS buffer, it won´t be harmed. The two buffer systems you
found are the two main procedures for hybridization. The Maniatis-type is
the older system and works well. The newer high-SDS based buffers also work
great, and are easy to prepare and cheap. I have routinely done my
chemiluminicent southerns in 5X SSC, 0,2%SDS, and 5% milk powder, and they
worked very well. For a biotin-based detection, however, NEVER use milk
powder (as it contains biotin), but use BSA or gelatine instead.
> or if not can someone direct me to
> a hybridization protocol compatible with chemiluminescence that doesn't
> require buying reagents? Is there a distinct advantage to buying
> hybridization buffers and blocking buffers?
For the company: Yes :)
For you: no!
Hope this helps,
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