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DNA Sequencing Gels

2024-10-11 DNA实验 加入收藏
Buffers and gel solutions Long Ranger: we started using this in early 1995. Grea

Buffers and gel solutions Long Ranger: we started using this in early 1995. Great stuff; the best thing is that the gels are not sticky after drying, even without removal of the urea. Long Ranger Gel Solution is available from many scientific supply companies and is a JT Baker reagent, cat # 4730-02 (50% concentrate).

 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 TBE5% in 1.2X TBE6% in 1X TBE
urea40.4 g40.4 g40.4 g
TBE13.3 ml 6X(8 ml 10X)16 ml 6X(9.6 ml 10X)13.3 ml 6X(8 ml 10X)
Long Ranger (50%)8 ml8 ml9.6 ml
water30.5 ml(35.8 ml)27.8 ml(34.2 ml)28.9 ml (34.2 ml)
ammonium persulfate70 mg70 mg70 mg

1. Mix to dissolve urea, filter through Whatman 3MM filter or equivalent.

2. Add 25 μl TEMED, and pour gel.

Electrophoresis

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℃.

3. A 10 min prerun is sufficient

4. Maintain gel temperature between 40-50℃.

5. Do not heat samples excessively during denaturation step. 2 min at 75℃ 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.

Electrophoresis Buffers (TBE)

10X (per liter)6X (per liter)
Tris base108 g64.8 g
Boric acid55 g33 g
disodium EDTA9.3 g5.58

(The TBE should be fresh-if the boric acid precipitates, make a fresh stock--we find that a 6x solution lasts longer before precipitating)


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