发信人: moso (野鬼), 信区: Biology
标 题: Re: 求助: Southern Hybridization with olig
发信站: Unknown Space - 未名空间 (Thu Aug 5 11:58:26 2004), 转信

I don't quite understand how you label the probe. Usually the methods for
making non-radioactive probe incorporate only a small unit, like a biotin,Dig
or fluorescein into the probe via UTP or others instead of directly incorporate
the enzymes for detection,like HRP, AP. Since it is not easy to keep the full
enzymetic activity during the long experiment procedure.
So if you use klenow to label the probe, you can deactivate the klenow by
heating the reaction at 75C for 20min in presence of 10mM EDTA. Then you can
add the probe after denaturing into the DNA for incubation.
Considering the volume, I am wandering if you can use a small volume for
labeling. Actually you don't need really a 300ng probe.
【 在 bigfatliar (Chico Verde) 的大作中提到: 】
: Thank you again for your thoughts! I was using a Alkline Phosphotase based
: labeling kit for the hybridization. Alk phos was crossed linked to the probe
: using the reagents the kit supplied.
: The volume will go up to 96 uL for 300 ng of the labeled probe. I am not sure
: a freeze concentration will work or not since there is no purification after
: labeling. Without a purification and with concentration, the leftover labeling
: reagents may be adverse to the hybridization. I am aslo worrying that the left
: over labeling reagent may label the genomic DNA itself, which will kill this
: nice design. If I do an ethanol pricipitation, however, the enzyme may be
: deactivated and detection will not work.
: The other thing I am worrying is that oligos will not bind as favorably as the
: complimentary genomic DNA band. Thus after denaturing and mixing together,
: maybe very little will bind in the aquous buffer. This may not be a problem
: since hybridization on the membrane is the same thing.
: But I guess I should try it first anyway. If you have any suggestions,
: critiques of what I said above, please let me know.
: Thanks much!
: : 1. digest the genomic DNA in 1 ml. Precipitate DNA with ethanol and resolve
: : the DNA in a proper volume.
: : 2. Denature the genomic DNA by heating it at 100C. You can try to place the
: : DNA on ice to keep the sing stranded status. Considering the volume, it
: may
: : not work very well. So it may be better to keep the DNA at 100C before
: : adding probe. (it may not be a good idea to let the DNA stay at 100C for
: : long time)
: : 3. At the meantime of denaturing the genomic DNA, label the probe( and
: denature
: : it if the oligo is not single standed).
: : 4. Add the probe and blocking DNA(single standed or denatured) to the
: denatured
: : genomic DNA and let the temperature go down slowly. Incubate the mixture
: at
: : room termperature ( /27C )for 1-3h.
: : 5. Run the gel,transfer the DNA to memberane and fix the DNA using a regular
: : protocol.
: : 6. Detect the probe.
: : For one thing you should give some concern. You need to optimize the
: : concentration of your block DNA, eg. random 16mer oligos, so that you can
: : acquire an ideal ratio of signal to noise.
: already
: hybridization
: sounds
: the
: done
: do
: that
: with


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