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), 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