dialysis of RNA

tfitzwater at NEXSTAR.COM tfitzwater at NEXSTAR.COM
Thu Sep 16 12:32:04 EST 1999


>From: carmelo.diprimo at bordeaux.inserm.fr (Carmelo)
>Subject: dialysis of RNA
>Date: 16 Sep 1999 11:06:45 GMT

>I would like to dialyze small RNAs (between 10 and 60 bases). I'm just
>wondering if the non sterile spectra/por membranes cellulose ester (used
>without futher preparations) are good for that. Can I consider these
>membranes are RNase free?

>Carmelo

This is my protocol for preparing RNase-free dialysis tubing.
Preparation of dialysis tubing for electroelution of nucleic acids

1.   Tare a 1000 mL glass beaker.
2.   Add 46.53 g Na2EDTA.
3.   Add 6.2 g NaOH pellets.
4.   Add 450 mL Type I water.  Stir.
5.   Add 10 g sodium bicarbonate.
6.   Add 1000 µL DEPC if preparing RNA grade tubing.
7.   Remove stir bar.  [Buffer = 250 mM EDTA/2% bicarbonate]
8.   Wear gloves and cut 18 mm flat width MW cutoff 3500 dialysis tubing to
12 to 18 inch segments using clean scissors wiped with 70% ethanol.
(Spectrapor 3 catalog number 32720).
9.   Do not overfill container.  Place 500 mL Erlenmeyer flask on top to
keep tubing submerged.
10.  Boil in microwave for 30 minutes on 30% power.  (The traditional
method of boiling over a flame in a fume hood produces more boilovers and a
strong scorched smell.)
11.  Clean up spills.
11.  Rinse in stream of Type I water into sink. Fill to check for
punctures.
12.  Transfer tubing to a wide mouth container filled with  50% ethanol
after rinse.  Avoid containers with sharp edges.
13.  Drain and replace 50% ethanol after all tubing prepared.
14.  Store 4°C.

Tim Fitzwater
NeXstar Pharmaceuticals (Gilead Sciences)




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