protocol to perform a RNA dot-blot?

Klaus.Matthaei at anu.edu.au Klaus.Matthaei at anu.edu.au
Fri Aug 27 04:05:36 EST 1993


>Hi,
>
>A friend is posting this question for me.
>I'd like to find a protocol to perform a RNA dot-blot, in which formaldehyde
>is replaced by something less toxic.
>
>I'm working as a Ph D student at the department of molecular biology at 
>the Agricultural University of Wageningen.
>
>My email-adress is: Heleen.Frings at mac.mb.wau.nl
>thanks in advance.
> Heleen Frings
>
>----------------------------------------------------------------
>Heleen Frings
>Department of molecular biology
>Agricultural University of Wageningen
>
>e-mail: Heleen.Frings at mac.mb.wau.nl
>----------------------------------------------------------------


Hi Heleen

Alkaline transfer to positively charged nylon membrane might get you there.

Both native and denatured RNA is quantitatively retained by PCMN
(positively charge-modified nylon).  RNA must be totally denatured and is
achieved by the following.

Immediately before use dissolve RNA in 0.5ml of ice-cold 10mM NaOH, 1mM EDTA.

Assemble dot blot apparatus with a sheet of Nylon (I use Hybond N+ but I
have no affiliation to Amersham)) prewetted thoroughly in water.

Apply samples (in serial dilutions in the same solvent if you want)

Apply vacuum until aspirated.

Apply 0.5ml cold 10mM NaOH, 1mM EDTA the vacuum to rinse.

Dissasemble and rinse membrane in 2 x PE, 0.1% SDS few minutes to neutralise.

Hybridise as normal.

Cheers and Good Luck

Klaus
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Klaus Matthaei
Gene Targeting
The John Curtin School of Medical Research
The Australian National University
E-mail: Klaus.Matthaei at anu.edu.au

"Think twice - Do once"
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