Lab Experiment 18.1: Resolve the mRNA isolated from the liver on the denaturating Agarose gel electrophoresis.
Background Information: The protocol for RNA gel is similar to the DNA gel discussed in the previous lecture except that the RNA gels are performed under denaturating conditions. RNA sample and the agarose gel contains formaldehyde to denature the secondary structure present in the RNA and prevent the re-formation of double stranded region in the RNA structure (formation of secondary structure affects the migration and resolution of RNA on agarose gel). Presence of secondary structure in RNA allows the faster migration of RNA on agarose gel. As a result, it gives less time for the molecule to interact with the agarose gel and consequently less resolution within the different RNA species. Destruction of secondary structures in the RNA structure minimizes these effects and allows better separation on agarose gel.
Materials:
1. Agarose, Molecular Biology Grade.
2. 10x MOPS Buffer:
Composition:
0.4M 3-(N-morpholino)-propanesulphonic acid (MOPS), pH 7.0
0.1M sodium acetate
10mM EDTA, pH 8.0
MOPS buffer can be stored upto 3 months at 40C. For preparing 1xMOPS buffer, it can be diluted with RNase free water.
3. 37% formaldehyde, pH> 4.0
4. RNA molecular weight ladder
5. 0.5M ammonium acetate
6. 0.5µg/ml ehidium bromide in 0.5M ammonium acetate
7. RNase Free H2O
8. Formamide
9. Formaldehyde loading buffer:
Composition:
1mM EDTA, pH 8.0
0.25% (w/v) bromophenol blue
0.25% (w/v) xylene cyanol FF
50% Glycerol.