My dot blot hybridization doesn't work! - (Sep/06/2007 )
I had been trying to do hybridization with nylon membrane for some times. I boiled the extracted RNA samples for 5 minutes in 100 degree C. Then spot 5 microlitre to the membrane at distance with other sample. After completely dried at RT, the membrane soaked in blotting buffer for 5 minutes and continue on with incubation for 15 minutes. After discarded the buffer, the membrane left to air dry..and UV for 5 minutes after dried completely.
Pre-hybridization solution was used and incubated with gentle agitation for 2 hrs..and continue with hybridization where 1 microgramme of the labelled probe added into the pre-hybridization solution. The setting left to be run for overnight. NExt day..post hybridization by washing it with low stringency buffer followed by high stringency buffer. After the, washing buffer used as to equilibrated the membrane and continue with the blocking buffer for 2 hrs. And continue on with DIG labelling following protocol by da manufacturer-Roche as the antibody solution added into the blocking buffer. The membrane then washed using washing buffer for twice and equilibrate by using dectection buffer. Then the colouring-NBT/BCIP added into the dection buffer as the colouring solution. The setting left for spot development and wrapped with alluminium foil and kept in dark.
I couldn't get any positive result although the background colouring was quite nice 
. I once do a sensitivity test to test out whether 1 microgramme labelled probe is sufficient..and it indeed shown positive spot. Another more question that bothering me..is will 1 microlitre of the labelled probe equal to 1 microgramme of the labelled probe?
If anyone out there can lend me some help...is very much appreciated..and I am desperately need some help. Thanks 
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The first thing you should do is to do serial dilutions of your probe, spot the dilutions on a membrane, and detect the spots with your detection methodology. This will tell you how sensitive your detection is, and how well labeled your probe is.
Second, do serial dilutions of a positive control sample containing your target, and again spot onto a membrane, crosslink, hybridize with your probe, and detect. This will tell you how sensitive your hybridization/detection is.
Only when this works should you look at your real samples. I would recommend doing both controls on every run, along with your real samples. At least you'll know where the problem is, and the limits and linearity of your detection.
Second, do serial dilutions of a positive control sample containing your target, and again spot onto a membrane, crosslink, hybridize with your probe, and detect. This will tell you how sensitive your hybridization/detection is.
Only when this works should you look at your real samples. I would recommend doing both controls on every run, along with your real samples. At least you'll know where the problem is, and the limits and linearity of your detection.
Thanks...
[quote name='phage434' date='Sep 6 2007, 08:51 PM' post='110879']
The first thing you should do is to do serial dilutions of your probe, spot the dilutions on a membrane, and detect the spots with your detection methodology. This will tell you how sensitive your detection is, and how well labeled your probe is.
I had done serial dilution to my probe and only the lowest diluted shown positive dot spot. Then I proceed with positive control and try to detect. But it didn't show any positive result even to the positive control. How am I going to do? Retest again until I got something positive shown up or try up other alternative..which I am hoping so. Thanks.
Well, doesn't this tell you that your probe is poorly labeled? Do you have a positive control for the probe? How are you making the probe? I have had very good luck making probes by spiking a normal PCR reaction (using Taq or a Taq+Pfu mixture) with dig-11-dUTP at 5-10% of the normal dTTP concentration. Taq is required because Pfu and other enzymes like it do not incorporate dUTP.
I am using DIG Oligonucleotide 3' - END Labeling Kit, 2nd Generation from Roche. The protocol given is add 100 pmol oligonucleotide and sterile double distilled water to a final volume of 10 micro litre to a reaction vial. Then Add in reagent according to the manufacturer protocol which are 5x Reaction buffer (vial 1) to final concentration of 1x, followed by CoCl2 (vial 2) to final concentration of 5 mM, next is the GIG-ddUTP solution (vial 3) to final concentration of 0.05 mM and lastly Terminal transferase (400 U, Vial 4) to final concentration of 20 U/ microlitre. Then mix and centrifuge briefly. before incubate at 37 degree celcius for 15 min and then place on ice immediately. After that, the reaction stopped by adding in 2 microlitre 0.2 M EDTA (ph8.0)Do you think that there is something amiss? Do tell me..thanks. And oh..by the way, how do you put all those symbols of unit e.g micro, degree celcius and etc? Thanks once again
. So, this is a 3' labeled oligo probe. You may have trouble binding the short oligo to the membrane. I'd recommend UV crosslinking the membrane after spotting dilutions to make sure the DNA stays put. Can you use a PCR probe instead? I've never tried the terminal transferase, but in principle it should work.
PCR probe? Well..I do have the UV crosslinking step mentioned after I boiled the sample in 100 degree celcius for 5 minutes before spotted onto the membrane and let it air dried. After the incubated in the blotting for 15 minutes, the membrane was put to air dried again before UV for 3-5 minutes. Do you think the UV duration is not enough? Me and my supervisor are now worried that the problem came from our probe. So now we are still trying. Hopefully some positive outcome observed. 
I don't know what you mean by "incubated in the blotting for 15 minutes". The crosslinking should happen right after the spotting (and perhaps dryiing). If you are washing the membrane before the crosslinking, then you are likely removing the DNA at that step.
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.
Hi.
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.
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