RNA isolation

Arnoud van Vliet avvliet at knoware.nl
Mon Nov 8 17:27:00 EST 1999


Posted on behalf of yoel shiboleth :

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Dear Arnoud
here's the protocol:
10X running MOPS buffer:
0.2M MOPS
50mM NaOAc
10mM EDTA pH8.0
H2O Ster to 1L
pH to7.0 with 10N NaOH

1.Prepare 50mM NaOAc with ster H2o (16.7ml/L from 3M NaOAc stock) or add
directly as powder.
2.Dissolve 41.86gr/L MOPS (Sigma or other molecular biology grade)
3.add 20ml/L EDTA pH 8.0 0.5M
4.adjust pH with 10M NaOH, and finaly with 2M to 7.0
5. adjust H2O to 1L
6. Filter sterilise (don't autoclave) w 0.2micron millipore under vacuum
Buffer keeps for very long in the fridge if kept dark (use allufoil) in
filter bottle.

MOPS Loading mix: (this mix is good for loading RNA on regular gels too at
about 2-3 mix : 1RNA ratio)
in hood:
10X MOPS 4ml
Formamide 20ml
Formaldehyde 0.7ml

aliqout to 2ml tubes and keep at -20C for ever (won't freeze)

Gel: (100ml)
melt agarose in 60ml H2O. Cover to eliminate evaporation but make sure it
boils.
In a chemical hood prepare gelbox and :
Combine in a cylinder 10X running MOPS 10ml, Formaldehyde to 0.22M (1.8ml
*37%/100ml), room temp H2O to 40ml (28.2ml). Pour into hot agarose, swirl
carefully to mix, add 1/2 the amount of EtBr you would usually use if you
want, and pour carefully in hood.

Running Buffer: (1L)
10X running MOPS buffer 100ml
Formaldehyde 18ml
H2O 882ml
Buffer keeps at room temp in dark bottle.

RNA sample: 10-20 ug (40ul, if you want more, keep mix ratio and make a
thicker gel)
RNA in H2O 15.3ul (conc 1.3ug/ul if you want 20ug in 40ul)
MOPS loading mix 24.7ul
Heat 10 minutes to 60C
Ice
add some "blue-juice" and load

Run at 75V or less for about 4 Hrs mixing on two magnetic stirrers or mix
buffers with an electric dispensor once an hour. This is critical to stop pH
from going basic at the anode (yeah, I know,  I made a mistake yesterday).
Check with pH paper.

If you added EtBr you can photo gel.
If you want to transfer large bands you should treat gel (especialy thick
ones) with 0.05N NaOH (1ml 10N +200ml H2O) for 15 minutes exactly prior to a
few water rinses, pyrex exchange, 20XSSC 45mins with a change in the middle.
If your blotting was good, a picture of the "pancake" will show nearly no
ribosomal RNA.

Please post this letter on the newsgroup, as I don't have access to it from
here.
Tot siens
Yoel







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