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Help with Northern Blotting


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#1 robsabba

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Posted 19 August 2009 - 02:03 PM

Hi

I am having problems with Northern Blotting of potato periderm RNA. I am using DIG labeled oligonucleotide probes and Roche's DIG Easy Hyb Granules. I believe the problem is with the hybridization, which I am doing overnight @ 50-60 C in tubes. I am also going to try using modified Church-type buffers instead of the Easy Hyb granules. Eventually, I wish to use these probes for ISH. I would appreciated any advice on this.

Thanks!

#2 bob1

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Posted 23 August 2009 - 04:37 PM

An explanation of the problem would be useful! Have you tried altering the hyb conditions -time and temperature? Have you checked that the hyb oven is running at the correct temperature?

#3 robsabba

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Posted 24 August 2009 - 06:45 AM

An explanation of the problem would be useful! Have you tried altering the hyb conditions -time and temperature? Have you checked that the hyb oven is running at the correct temperature?

Thanks for the reply. Yes, I have changed hybridization temperatures (between 45 and 60C). I have mostly been hybridizing overnight to get as much hybridization as possible. In particular I was wondering if anyone has experience with Roche's DIG Easy Hyb Granules and if they experienced any problems with it. Since they don't say what is in their product (other than it does not contain formamide) there is no good way of knowing how to alter the buffer itself (ionic strength, etc.). Unless, perhaps others have had better results adding something like formamide to the product.

I have started using modified a Church type buffer now, so that I can add or subtract BSA, formamide, dextran sulfate, PEG and alter salt concentrations, etc.

#4 phage434

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Posted 24 August 2009 - 05:51 PM

First check that you can detect your probe by spotting 10x dilutions of the probe on a membrane and checking detection. Note the detection limit. Then, spot your positive sample in 10x dilutions and detect with your probe. Note the sensitivity. Only when this is working well should you bother blotting anything. It is far easier to optimize spotted DNA than a blot.

#5 robsabba

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Posted 25 August 2009 - 06:40 AM

First check that you can detect your probe by spotting 10x dilutions of the probe on a membrane and checking detection. Note the detection limit. Then, spot your positive sample in 10x dilutions and detect with your probe. Note the sensitivity. Only when this is working well should you bother blotting anything. It is far easier to optimize spotted DNA than a blot.

Thanks for the reply. I have tested the probes by blotting directly to nylon (in a dilution series) and I get good detection with the antibody. I have tried blotting RNA from tissue (in a dilution series) which is expressing high levels of the target mRNA directly to nylon and then probing it. However, I have gotten false positives with this (as well as no labeling at all) and I have been told it is not a very reliable method. I am also trying probes for the r25s mRNA, as a positive control, as it is very highly expressed.




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