My dot blot hybridization doesn't work! - (Sep/06/2007 )
You should not have to boil the probe at 100C if it is an oligo. The point of the boiling is to denature double stranded DNA, and the oligos are already single stranded. I would think the UV exposure is long enough. Is the exposure done with a crosslinker, or with a standard UV transilluminator? The UV should be short wave, 254 nm if available, 302 if not, but 365 nm will probably not do much.
Well..it started to make sense that you are right. Incubating it in a blotting buffer is from a previous protocol by a previous student. Suprisingly, the student do have the positive spot shown after dectection. I used the same protocol, and I didn't get anything. I think your suggestion of doing crosslinking after the drying is good. I will try that to make sure I crosslinking before I do anyhing further. The UV I am using is in the range of 300-360 nm. I will also try to change it to a shorter wavelength. Thanks for the help.
The boiling point....is another matter that borther me. Maybe I will try as suggested by BIOwizard that using a lower temperature such as 65.
Thanks for the help. I will try it out as soon as possible. Hope that the reagents that I have are enough for me to try.
Common prob dealing with RNA is that it's easily degraded. I quess boiling at 100 C, 5' will not affect the integrity ot RNA but I'd prefer to lower it to say... 65C. Also, Have to really make sure that everything is RNase-free.
Have you tested the specificity of the oligonucleotide(s) to the target RNA? E.g. on Northern or by other means? Is the oligonucleotide a ds or ss? If ds, no worry, if ss.. do make sure it's the antisense sequence.
If you're using Nylon Plus, UV crosslinking is not really necessary, though I'd go with it anyway. But I don't think that's the prob.
I'm more concerned with the "specificity" of the oligonucleotide with the target RNA and also the integrity of RNA.
...-...
I am indeed using Nylon Plus membrane. So do u think that if I put it into a blotting buffer before I UV to crosslinking it will wash out all the RNA that spotted onto the membrane? The boiling point that I am using is from a previous student protocol. Am I am now in concern of the amount of RNA that I spotted onto the membrane is not sufficient enough. Suggestion by my supervisor is to check out from range of 1microgramme to 5 microgramme and check which one is strongly probed by the labeled probe. What do you mean the oligonucleotide is whether ds or ss? The oligonucleotide of my probe or my sample? According to the briefing by my supervisor, the oligonuleotide of the probe is ss because it will then bind to the sample spotted onto the membrane. I am not so sure about that since i only know that the sample is RNA and proven to work by previous student. 
The probe labelling efficiency test had been done before in a serial dilution and the undiluted is the positively bind and shown spot. As for the specificity of the probe, it was according to a reference journal where the probe is said to be positively bind to the reference sample. I didn't know whether you understand what I am trying to say..but hopefully someone can help me. I am now desperately need help. Thanks.
> I'd do that.
> ok. Will have to trust the previous person working on it and journal then...
You've tried positive control and got nothing.... you've tried spotting the labelled oligonucleotide and you could detect the signal from lowest dilution. Then it could really be the RNA prep... if your oligonucleotide do bind to genomic DNA of your sample, you could use gDNA as your another positive control among the RNAs. If the gDNA lights up while RNA don't, then you'll have to make sure your RNA is alright (not degraded or too diluted to be detected with your probe).
...-...
I will try to work it out firstly on the serial concentration to be spotted onto the membrane. As the point brought out by Phage that the uv crosslinking should be done before blotting coming to make sense. I had tried asking my supervisor about it and we do agreed that Phage is correct. It seems to be suspecious how the previous student got it...but..well, it doesn't matter for me, as long as I got the right method by the help and guidance of all to do the experiment and research...it's all right. Thanks. I will start to try it out as soon as possible. Really thanks for the help. 
Hi all,
Thank god and thanks to all of you who had help into solving the hybridization problem. It seems that all those suggestion by all of you had brought of some positive outcome. The probe labeling efficiency test shown really good result and later, I shall proceed with the hybridization for the samples. Once again, thank you. 
Hi all,
This is me again, and in this time I was wondering how do you use your recycle hyb solution, or you didn't reuse the hyb solution? I haven't try out to use the recycled hyb solution. All that is bothering me is that do you thaw it completely on ice before using it or you will thaw it and pre-heated it in the required temperature or you straightly pre-heat it in the required temperature? And I am thinking, if I am doing lebeling efficiency test, do I really need to pre-hyb and hyb it for the membrane that had been spotted with the probes?
Hi cheerioet83,
I found this nice link from Roche a few minutes ago.
Although it doesn't say anything about the reuse, it might be interesting for you...?
https://www.roche-applied-science.com/prodi...MAN/dig_toc.htm
Greetings (once again ),
Chakchel
I found this nice link from Roche a few minutes ago.
Although it doesn't say anything about the reuse, it might be interesting for you...?
https://www.roche-applied-science.com/prodi...MAN/dig_toc.htm
Greetings (once again
),Chakchel
Yo...
,I do browse through the website that is given by you. I still couldn't get the point of how to reuse it..but I will still be searching for it. Thanks for the help. Indeed I had been helped after I had posted this topic. Thanks to all of you..I can continue back my hybridization.
Cheers
For spotted probe detection, you don't have to hybridize at all. What is there to hybridize? You just add the anti-DIG-enzyme conjugate and go. I don't believe it is common (or a good idea) to try to reuse that solution. Antibody reuse is common if you are doing a series of westerns with a rare primary antibody which you are using a secondary antibody to detect. Conjugated antibodies such as the anti-dig need to remain enzymatically active as well as bind to their antigen. While it might work to recycle them, it would not be the first thing I'd try. I would probably try to store them at +4 rather than freeze them, and perhaps add small amounts of an antibacterial agent (thiomerisal, e.g.). A different thread claims that sodium azide is an inhibitor of HRP enzyme action, so I'd avoid that.
Erm...yaya.. you are right. There is no need to hyb. The antibody solution that I use is Anti-Digoxenin-AP conjugate (750 U/ml) from Roche; where I added in 6 microlitre of Anti-Dig into 30 ml of blocking solution (Skimmed milk + Maleic Acid). Is it possible to store the antibody solution after I used it? Well, for the hyb solution, I am still wondering and wanted to try out as my supervisor said it can be reuse up to 3 times. I am still working on it. And if I can get anything from reusing the hyb solution I will definitely post it up. I am now on my way to test out the efficiency of the needed probe. I guess I might take another few days to do the hybridization by using the recycled hyb solution. I am indeed storing the Anti-Dig in 4 degree celcius. Haven't try with adding antibacterial agent. Why do we need that anyway? Thanks for helping.