MOPS RNA running buffer

Arnoud van Vliet avvliet at knoware.nl
Thu Jun 22 15:32:06 EST 2000


> Con anyone out there tell me how to stop my MOPS buffer turning yellow
> after I have autoclaved it?

Colin, you can't. But there is an alternative; that is to use sodium
phosphate buffer. I have switched to a 20 mM NaP buffer, which is easy to
DEPC-treat, autoclave etc, and also very easy to make. Only problem: with
its high ionic strength, a lot of current is going through the gel already
at low voltage, so if you run it at 50V at an environmental temp of >30
degrees C WITHOUT cooling, then it melts. Unfortunately I tried that this
week during the heatwave in the Netherlands (34 C). So cooling might be
necessary or slow running (and some patience)

The protocol for the gel is attached at the end of this mail. Feel free to
contact me if you need more information.

Hope this helps,

Arnoud



PREPARATION AND RUNNING OF FORMALDEHYDE-AGAROSE GELS (1.5%)

1. Make tray, comb and tank RNAse-free by cleaning with soap, washing with
demi-water, and  incubating for at least 30 min in 0.1-0.2 N NaOH, followed
by washing with RNAse-free water. Dry tray and comb, when necessary briefly
clean with 70% EtOH
2. Pour gel:

for: 100 ml (midi gel, max 20 lanes with 2 combs)
75 ml 2% agarose (cooled to 60-65 degrees C)
2 ml 50x Running buffer
5.3 ml formaldehyde 37%
17.7 ml aqua dest, RNAse-free
Pour directly after mixing, in fumehood

Let set for minimal 30 min, put wrap over gel. Formaldehyde gels are not as
strong as corresponding normal agarose gels!

3. Make Running Buffer: dilute 50x Running buffer in RNAse-free water.
Volume needed:

4. Prepare RNA samples:
Mix 2 ml RNA sample with 8 ml RNA denaturation mix
Incubate at 65°C for 15 min
Add 1 ml loading RNA loading buffer

5. Put gel in tank, submerge in buffer. Careful with slots, as gel is weaker
than normal agarose gel. Submerge in as little buffer as possible
(IMPORTANT!)

6. Load samples, and run gel at less than 50V until bromophenol blue band
reaches has run 3/4 of the gel.

Materials

50x Running buffer
0.9 M Na2HPO4
0.1 M NaH2PO4
(Optional: Treat with DEPC)
Autoclave

2% Agarose
2 g agarose in RNAse-free aqua dest
Autoclave

10x RNA loading buffer
50% Glycerol
1 mM EDTA
0.25% Bromophenol blue (Sigma)
0.25% Xylene cyanol (Sigma)
Treat with DEPC, autoclave

RNA denaturation mix (per 2 ml RNA sample)
0.16 µl 50´ Running buffer
1.75 µl formaldehyde 37%
5 µl formamide, deionized
1.09 µl RNAse free water

For 20 samples of 2 ml:
3.2 µl 50´ Running buffer
35 µl formaldehyde 37%
100 µl formamide, deionized
21.8 µl RNAse free water









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