RNA Formaldehyde GELS
1. Pouring the gel
Add 1.2 g agarose to 72 ml ddH2O. Melt. Cool with gentle heat and stirring. Add
10 ml of 10X MOPS/EDTA and 18 ml of 37% formaldehyde (in fume cupboard). Pour
the Gel.
10X MOPS/EDTA
0.2 M MOPS
0.05 M NaOAc
0.01 M EDTA
41.86 g
4.10 g
3.72 g
______
PH to 7 w/ NaOH
ddH2O to 1 L
2. Sample preparation
RNA
Formamide (Deionised)
37% Formaldehyde
10X MOPS/EDTA
Bromophenol Blue
EtBr (400ng/uL)
Per Sample
Markers+
_______________________
5ul#
5ul
10
10
3
3
2
2
2
2
1
1
_______________________
23ul*
23ul
# If you use less than 5ul of RNA, make up the difference with water.
+ The markers are from BRL catalog number 5620SA. It is a 0.24-9.5 Kb ladder (1
ug/ul).
* You can increase resolution by decreasing the total volume of your samples to say 10
ul and pouring a thinner gel.
Heat the samples at 65 C for 15 minutes. Chill on ice 2 minutes. Spin down. Load.
3. Running the gel
Run the gel at 70 Volts using 1X MOPS/EDTA as the running buffer.
Alternatively, if you are worried about the formaldehyde diffusing from the gel during
electrophoresis, the running buffer can be supplemented with 185 ml of 37 %
formaldehyde per 815 ml of 1X MOPS/EDTA. Recirculate the buffer with Hybaid
"Buffer Puffer" tank. You may want to run the gel in the cold.
Buffer:
100 ml 10 x MOPS/EDTA
900 ml ddH2O