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 (Wed Aug 4 18:45:28 2004), 转信

Whether or not the probe is excessive basically depends on the level of the
targetting DNA. Generally I believe the concentration of your probe should be
enough for the detecting because 10ng/ml of a 16mer oligo aproximately equals
to 1.5nM by molar concentration, which could capture 1.5pmol targetting DNA if
it bound at a ratio of 1 to 1.
To label or not to label, it depends on the method you used to detect the
targetting. If you use, for example, biotin to lable to probe, then label the
probe before incubate it with digested genomic DNA. Run the mixture on an
agarose gel after incubation. Follow the normal protocol to blot and fix DNA.
Now the membrane is ready for detection.
I don't know what method you used for labelling. I normally have a 50ul probe
after label the DNA with 32-P. We can discuss it afterwards.
I don't have a ready to use protocol. Now,I give an idea. See if it is
feasible to you.
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.
【 在 bigfatliar (Chico Verde) 的大作中提到: 】
: Thank you for the suggestion. I am not sure what you meant that I have already
: involved excessive probe. The concentration of the probe in my hybridization
: buffer is only 10ng/mL.
: In your suggstion, I should hybridize the probe (labeled, unlabeled? It sounds
: like the labeled.) with the genomic, then run the gel? I then blot it to the
: membrane and detect it right away? This sounds a nice idea. Has anyone done
: this before? For labeling my probe, the volume will go up a lot. Also, I do
: need to denature my DNA before the hybridization, don't I? Do you have a
: related protocol?
: Waiting for your reply and thank you for your ideas!
: 【 在 moso (野鬼) 的大作中提到: 】
: : I did not do such kind of southern before. To my knowledge, it seems that
: the
: : probe is not very competitive if the probe does have a good homology with
: your
: : genomic DNA because the length of probe is too short to gain a priority,
: : though you have already involved excessive probe in the hybridization
: reaction.
: : I think, maybe you can try to make a hybridization before blotting, like the
: : procedure of labeling the probe by nick translation, letting the random
: primers
: : bind to the denatured template before klenow starts to work. To your case,
: you
: : can run the DNA after a certain time of incubation, then transfer DNA using
: the
: : normal procedure. Detection can be carried out right after the blotting.
: : Good luck and Welcome to further discussion.
: appreciate


--
※ 来源:．Unknown Space - 未名空间 mitbbs.com．[FROM: 165.230.]