DNA Sequencing Gels

Buffers and gel solutions

The table below is used by our lab, and is sufficient for a standard 35 x 43 cm x 0.4 mm gel (we use the Owl boxes). The Long Ranger solution comes with a troubleshooting guide; a few of the electrophoresis parameters that have been demonstrated important for us are listed below.

Long Ranger Gels

	5% in 1X TBE	5% in 1.2X TBE	6% in 1X TBE
urea	40.4 g	40.4 g	40.4 g
TBE	13.3 ml 6X (8 ml 10X)	16 ml 6X (9.6 ml 10X)	13.3 ml 6X (8 ml 10X)
Long Ranger (50%)	8 ml	8 ml	9.6 ml
water	30.5 ml (35.8 ml)	27.8 ml (34.2 ml)	28.9 ml (34.2 ml)
ammonium persulfate	70 mg	70 mg	70 mg

1. Mix to dissolve urea, filter through Whatman 3MM filter or equivalent.
2. Add 25 ul TEMED, and pour gel.

1. For maximum base reads, use 1.2X TBE in the gel and 0.6X TBE as the electrophoresis buffer.
2. Run gels at 55 W. Wattage must not exceed 55-60 W even with large 40 x 40 cm plates or a temperature of 50 C.
3. A 10 min prerun is sufficient
4. Maintain gel temperature between 40-50 C.
5. Do not heat samples excessively during denaturation step. 2 min at 75 C is enough, and it is not necessary to heat again prior to a second or third loading.
6. We have had the best results with 4.5 and 2 hour runs, at 55 W.

Older (pre-Long Ranger Gels) Method

164.0 g Tris-OH

27.5 g Boric Acid

7.45 g disodium EDTA

water to 1 l

Acrylamide stock

38% Acrylamide

2% bis-acrylamide

Gels (volume is for standard size sequencing gels)

8% gel:

40.4 g urea

27.0 ml water

16.8 ml 38/2 acrylamide

8.0 ml EB

6% gel:

40.4 g urea

31.2 ml water

12.6 ml 38/2 acrylamide

8.0 ml EB

5% gel:

40.4 g urea

33.5 ml water

10.5 ml 38/2 acrylamide

8.0 ml EB

Dissolve by heating slightly, degas if necessary. Add 0.7 ml 10% ammonium persulfate, and 25 ul TEMED, and pour gel immediately.