northern blot probe preparation - (Dec/30/2004 )

hi everyone

Can anyone please tell me how to prepare probe( radioactive or nonradioactive) for northern blot.

Also, which probe is better to prepare- RNA or DNA

thanks
gayle

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Hi gayle,

I don't know which type of probe you want to use. Basically there are three types of probe: double-stranded DNA, signel-stranded DNA (including oligos) and single stranded RNA. Each type can either be radiolabeled or nonradiolabeled. The best probe is RNA probe in terms of sensitivity.

RNA probes can be generated by in vitro transcription from DNA templates.
dsDNA probes can be made by RT-PCR from cDNA templates.
Single-stranded DNA probes can be made by primer extension, asymmetric PCR or end-labeling of oligos.

Design considerations:

Probes must contain antisense sequence to the target RNA
Length is usually 100-1000 nt, oligo probe should be at least 15 nt.
Minimual mismatch to other gene sequence.

Hope that helps.

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Hi,boy,
as fpr how to prepare the probe for Northern blot. i suggest you look for some information for Molecular cloning III.
talking about which one is better for Northern, nonradioactive is ok for Northern, but not for Southern. Radioactive is ok both Northern and southern.

nice to meet you,

Yours sincerely,
newdragon

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hi

Thanks a lot for your suggestion
is it ok to convert RNA to cDNA and then do a southern blot to analyze the presence of alternatively spliced forms of RNA

thanks in advance
gayle

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even if you probe RNA with cDNA it's still called a northern - not a southern. I incorporate alpha-32P-dCTP using the Prime-a-Gene® labelling system from Promega. works really well and with high labelling efficiency...

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hi

Thanks for your suggestion
I have bought random primer labelling kit from ambion
I meant to say by telling cDNA is that If convert my RNA to cDNA and run a gel and then hybridize it with a cDNA probe, will i be able to detect alternatively spliced form of RNA.

Thanks,
gayle

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