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northern prob


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4 replies to this topic

#1 michalcn

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Posted 28 January 2010 - 01:45 PM

Hi,
I'm trying to figure out how to design a northern blot, I have DNA in Topo TA cloning vector with T7 promoter so what is the next step should I use Prime a Gene to label it and I don't really understand the non label - how do you detect the hybridization?

Thanks for helping

Michal

#2 bob1

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Posted 28 January 2010 - 03:07 PM

Have a look at the Roche manual for filter hybrization .

#3 michalcn

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Posted 29 January 2010 - 09:42 AM

I Looked at the manual which is actually talks specific about DIG labeling and since I don't have any experience I'm willing to try it but I can't understand what facilities do I need to develop the mambran just film or specific system with specific filters?
and another thing is this system preferred to the fluoresence or the biotin?

thanks

#4 bob1

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Posted 31 January 2010 - 03:17 PM

DIG is probably the most common non-radioactive method of doing nucleic acid hybridisation... I wouldn't recommend using radioactive compounds unless you have worked with them and are set up for it.

If you use the luminescence method (alkaline phosphatase with CDP) then you need standard radiographic film (I use kodak x-omat bt) or a camera set up for taking pictures of luminescence. You can also detect using chromogenic substrates (NBT/BCIP) which will give a coloured band on your membrane, but is slightly less sensitive than luminescence. I haven't used a fluorescence method, so I can't comment.

#5 michalcn

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Posted 03 February 2010 - 09:51 AM

Thanks for your help
I decided to use the biotin as nonisotopic labeling and I bought the whole system from ABI with the MAXIscript and so on but now I found out that they have template for housekeeping genes (GAPDH, b-actin) only for mice and not for human. Do you know if there are commertial templates for human or do I need to design myself?
Michal




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