Bio-Rad Sequi-Gen GT Sequencing Cell - Manual (Page 36)

5. Ammonium Persulfate, 25% stock solution: 0.25 g in 1 ml distilled H2O (in a microfuge
tube). Make fresh daily (see Section 6.2).
6. A constant power (or constant voltage) power supply (see Section 6.3).
7. Slab gel dryer(see Section 6.3).
8. Table top micro-centrifuge
9. Gel loading syringe (e.g. Hamilton 701-SN, 28 Gauge, 1.25 inch needle)
10. 1.5 ml microcentrifuge tubes (see Section 6.5)
11. Adjustable pipettors (e.g. Pipetman P-20, P-200, P-1000)
12. Balance
13. Plastic wrap
14. Pipette tips, autoclaved (see Section 6.5)
15. Waterbath or Temp-Block at 95 °C.
16. X-ray film and cassettes (dark-room facilities)
17. Filter Paper (see Section 6.3)
18. Siliconizing solution or glass coating solution
19. Geiger Counter
20. Ice bucket
7.2 Standard Gel Protocol
The following protocol is for a standard 7 M urea, 5% polyacrylamide gel for DNA
sequencing. See Section 4 for additional information on gel casting, sample loading, and gel
electrophoresis. For ordering information on gel reagent and electrophoresis buffers see
Section 6.
1. Combine 63 g of urea, 15 ml of 10x TBE, and 25 ml of 30% acrylamide stock solution.
Bring the volume to 150 ml with distilled water (low heat may be required to dissolve the
urea, but do not boil).
2. Filter the solution through a 0.45 micron mesh filter (optional). Then, degas under strong
vacuum 5–15 minutes to remove dissolved oxygen.
3. Add 150 µl TEMED and 150 µl 25% ammonium persulfate (or one microliter of each
reagent for every milliliter of gel solution) prior to gel casting.
4. Cast the gel according to procedures in Section 4.
Note: Wedge spacers (see Section 6.1) increase the number of readable bases per lane in a
sequencing gel. The use of wedge spacers results in a gel which becomes gradually thicker
toward the bottom. As thickness increases, resistance, voltage, and DNA mobility decrease.
The resulting gel has bands more closely spaced at the bottom. Wedge spacers allow the use
of standard polyacrylamide solution and buffers. No alterations to the gel solution, gel
casting or electrophoresis protocols are required to run DNA sequencing wedge gels.
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