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DNA (RNA) or Protein Electrophoresis

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; Ficoll 400: A2252, AppliChem), 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).


Table 1 Tris-HEPES buffer.
Tris-base (mM), pK=8.2HEPES-acid (mM), pK=7.55 Tris/HEPES ration pH (20ºC)
6020 3/18.79
5620 14/58.75
5020 5/28.86
4420 11/58.60
4020 28.54
3420 17/108.43
3020 3/28.34
2620 13/108.24
2220 11/108.12
2020 18.05
2022 10/117.98
2026 10/137.85
2030 2/37.74
2034 10/177.64
2040 1/27.53
2044 5/117.46
2050 2/57.73
2056 5/147.30
2060 1/37.25


Table 2 Tris-MOPS buffer.
Tris-base (mM), pK=8.2MOPS-acid (mM), pK=7.28 Tris/MOPS ration pH (20ºC)
6020 3/18.74
5620 14/58.70
5020 5/28.62
4420 11/58.53
4020 28.46
3420 17/108.33
3020 3/28.22
2620 13/108.08
2220 11/107.91
2020 17.83
2022 10/117.73
2026 10/137.56
2030 2/37.43
2034 10/177.32
2040 1/27.18
2044 5/117.11
2050 2/57.01
2056 5/146.93
2060 1/36.89


Table 3 Tris-MES buffer.
Tris-base (mM), pK=8.2MES-acid (mM), pK=6.16 Tris/MES ration pH (20ºC)
6020 3/18.76
5620 14/58.72
5020 5/28.64
4420 11/58.54
4020 28.47
3420 17/108.32
3020 3/28.19
2620 13/108.01
2220 11/107.72
2020 17.50
2022 10/117.22
2026 10/136.80
2030 2/36.56
2034 10/176.39
2040 1/26.22
2044 5/116.13
2050 2/56.02
2056 5/145.93
2060 1/35.88

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