Rna gel

Thibaud Le Mouel lemouel at antibes.inra.fr
Fri Nov 29 04:40:56 EST 1996


>Can someone give a good recipe for a formaldehyde/agarose gel for rna. I
>see all kinds of different concentrations etc.. Does someone have one,
>that works consistantly. ALso I put etbr in my gels, is it better to
>stain later or in the gel. I know it decreases signal intensity, but I
>like seeing my rna. THanks
>
>--
>William Wu
>PhD student
>McGill University
>Dept of Experimental Surgery
>Shriner's Hospital For Crippled Children
>1529 Cedar Ave.
>Mtl. PQ. Canada
>H3G 1A6
>http://www.geocities.com/yosemite/1156/index.html


This is my protocol wich works very well.


     -RNA Gel :

        -0,9 g of agarose + 80 ml water : dissolution
        -When at 60=B0C Add 10 ml 10X running buffer + 10 ml formaldehyde

     -Running buffer :

        800 ml water
        + 100 ml running buffer
        + 100 ml formaldehyde

     -Samples :

           RNA in water                    RNA in formamide
           6 =B5l RNA at 5 =B5g/=B5l             10 =B5l RNA at 3 =B5g/=B5l
           + 2 =B5l formaldehyde             + 2 =B5l formaldehyde
           + 10 =B5l formamide               + 6 =B5l water
        -Put at 70=B0C during 2 min and put quickly on ice
        -Add 2 =B5l of loading buffer/etbr

     -Coloration :

        -Add at the last moment 1/10 etbr in loading buffer

     Running buffer :
        Na2HPO4                 (61 mM)         10,858 g
        NaH2PO4                 (39 mM)         6,080 g
        EDTA 0,5 M              (20 mM)         40 ml
        Eau                                     ---> 1 litre

     Loading buffer :
        Phosphate buffer 0,2 M  (0,1 M)         5 ml
        EDTA 0,5 M              (10 mM)         200 =B5l
        Glyc=E9rol 50%            (25%)           5 ml
        Bromophenol blue                        10 mg


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   (\__/)  .~    ~.)) *********************************************
   /O O  ./      .'   * Th. Le Mouel  <lemouel at antibes.inra.fr.>  *
  {O__,   \    {      * I.N.R.A.                                  *
    / .  . )    \     * 123, bld F. Meilland                      *
    |-| '-' \    } )) * B.P. 2078                                 *
   .(   _(   )_.'     * 06606 ANTIBES CEDEX    FRANCE             *
  '---.~_ _ _&        *********************************************
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