DNA/RNA analysis on non-denaturing agarose (or PAAG) gel electrophoresis
The following gel electrophoresis conditions are recommended:
- use 1x THE buffer (without DEPC-treated water, RNA/DNA can not degrade during electrophoresis)
- use agarose gel in the concentration of 1.0%-1.5%
- add ethidium bromide (EtBr) to the gel
- wear gloves to protect a hand contact with EtBr
The first sign of RNA degradation on the non-denaturing gel is a slight smear starting from the rRNA bands and extending to the area of shorter fragments.
The following characteristics indicate successful RNA preparation:
- For mammalian total RNA, two intensive bands at approximately 4.5 and 1.9 kb should be observed against a light smear. These bands represent 28S and 18S rRNA. The ratio of intensities of these bands should be about 1.5-2.5:1. Intact mammalian poly (A)+ RNA appears as a smear sized from 0.1 to 4-7 (or more) kb with faint 28S and 18S rRNA bands.
- In the case of RNA from non-mammalian sources (plants, insects, yeast, amphibians), the normal mRNA smear on the non-denaturing agarose gel may not exceed 2-3 kb. Moreover, the overwhelming majority of invertebrates have 28s rRNA with a so-called "hidden break".
In some organisms the interaction between the parts of 28s rRNA is rather weak, so the total RNA preparation exhibits a single 18s-like rRNA band even on a non-denaturing gel. In other species the 28s rRNA is more robust, so it is still visible as a second band.
Loading buffer (10x):
20% (w/w) Polysucrose 400 (
P7798, Sigma-Aldrich), 100 mM Tris-HCl (pH 8.0), 5 mM EDTA, ~0.01% (w/w) Orange G and ~0.01% Xylene Cyanol FF
Electrophoresis buffer (1x final concentration):
10xTHE:
200 mM Tris-OH, 200 mM HEPES, 5 mM EDTA, pH 8.06 (24.23 g Tris-base, 47.66 g HEPES (free acid), 10 ml 0.5 M EDTA, dissolve in MQ-water; bring final volume to 1 litre).
10xTME:
200 mM Tris-OH, 200 mM MOPS, 5 mM EDTA, pH 7.83 (24.23 g Tris-base, 44.85 g MOPS (free acid), 10 ml 0.5 M EDTA, dissolve in MQ-water; bring final volume to 1 litre).
10xTME:
200 mM Tris-OH, 200 mM MES, 5 mM EDTA, pH 7.5 (24.23 g Tris-base, 39.04 g MES (free acid), 10 ml 0.5 M EDTA, dissolve in MQ-water; bring final volume to 1 litre).
Tris-HEPES buffer
Tris-base (mM), pK=8.2 | HEPES-acid (mM), pK=7.55 | Tris/HEPES ration | pH (20ºC) |
350 | 50 | 7/1 | 9.08 |
300 | 50 | 6/1 | 9.01 |
250 | 50 | 5/1 | 8.93 |
200 | 50 | 4/1 | 8.82 |
150 | 50 | 3/1 | 8.68 |
100 | 50 | 2/1 | 8.46 |
50 | 50 | 1 | 8.06 |
50 | 100 | 1/2 | 7.56 |
50 | 150 | 1/3 | 7.30 |
50 | 200 | 1/4 | 7.13 |
50 | 250 | 1/5 | 7.00 |
50 | 300 | 1/6 | 6.90 |
50 | 350 | 1/7 | 6.80 |
Tris-MOPS buffer
Tris-base (mM), pK=8.2 | MOPS-acid (mM), pK=7.28 | Tris/MOPS ration | pH (20ºC) |
60 | 20 | 3/1 | 8.74 |
50 | 20 | 5/2 | 8.62 |
40 | 20 | 2/1 | 8.46 |
30 | 20 | 3/2 | 8.22 |
20 | 20 | 1 | 7.83 |
20 | 30 | 2/3 | 7.43 |
20 | 40 | 1/2 | 7.18 |
20 | 50 | 2/5 | 7.01 |
20 | 60 | 1/3 | 6.89 |
Tris-MES buffer
Tris-base (mM), pK=8.2 | MES-acid (mM), pK=6.16 | Tris/MES ration | pH (20ºC) |
60 | 20 | 3/1 | 8.76 |
50 | 20 | 5/2 | 8.64 |
40 | 20 | 2/1 | 8.47 |
30 | 20 | 3/2 | 8.19 |
20 | 20 | 1 | 7.50 |
20 | 30 | 2/3 | 6.56 |
20 | 40 | 1/2 | 6.22 |
20 | 50 | 2/5 | 6.02 |
20 | 60 | 1/3 | 5.88 |