PrimerDigital

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=8.2	HEPES-acid (mM), pK=7.55	Tris/HEPES ration	pH (20ºC)
60	20	3/1	8.79
56	20	14/5	8.75
50	20	5/2	8.86
44	20	11/5	8.60
40	20	2	8.54
34	20	17/10	8.43
30	20	3/2	8.34
26	20	13/10	8.24
22	20	11/10	8.12
20	20	1	8.05
20	22	10/11	7.98
20	26	10/13	7.85
20	30	2/3	7.74
20	34	10/17	7.64
20	40	1/2	7.53
20	44	5/11	7.46
20	50	2/5	7.73
20	56	5/14	7.30
20	60	1/3	7.25

Table 2 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
56	20	14/5	8.70
50	20	5/2	8.62
44	20	11/5	8.53
40	20	2	8.46
34	20	17/10	8.33
30	20	3/2	8.22
26	20	13/10	8.08
22	20	11/10	7.91
20	20	1	7.83
20	22	10/11	7.73
20	26	10/13	7.56
20	30	2/3	7.43
20	34	10/17	7.32
20	40	1/2	7.18
20	44	5/11	7.11
20	50	2/5	7.01
20	56	5/14	6.93
20	60	1/3	6.89

Table 3 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
56	20	14/5	8.72
50	20	5/2	8.64
44	20	11/5	8.54
40	20	2	8.47
34	20	17/10	8.32
30	20	3/2	8.19
26	20	13/10	8.01
22	20	11/10	7.72
20	20	1	7.50
20	22	10/11	7.22
20	26	10/13	6.80
20	30	2/3	6.56
20	34	10/17	6.39
20	40	1/2	6.22
20	44	5/11	6.13
20	50	2/5	6.02
20	56	5/14	5.93
20	60	1/3	5.88