The main advantage of this protocol is that it uses ethidium
bromide staining, instead of the more time-consuming silver nitrate
procedure. A special apparatus with one plate transparent to UV
allows the gel to be photographed without removing it from the
sandwich assembly. Additionally, the gel can be re-used up to four
Equipment: Mega-Gel Dual
High-Throughput Vertical Electrophoresis Unit from CBS Scientific.
(http://www.cbssci.com/). Catalog #:
Gel mix (for 6% non denaturing PAGE):
- 22.5ml 40% Acrylamide-Bis (29:1)
- 7.5ml 10X TBE
- Adjust volume with deionized water to 150 ml
- Just before use add:
- 1 ml 10% APS solution
- 110 µL TEMED
- Casting the gel: pour the gel directly into the plates
in vertical position. Avoid the formation of air bubbles. When
the volume is almost full, place the comb and clamp it to both
plates (do not push the comb). Finish pouring the rest of the gel
using a large syringe. Let it solidify for 60 min.
- Pre-running: Pre run for 45-60 min at 350V with running
buffer (0.5X TBE). Add approx. 20 µL of 10mg/ml ethidium bromide
to the buffer in the lower tank.
- Loading samples: Add 4 µL of 6X loading buffer (1) for
25 µL PCR reaction. Load 15 µL of sample per well. Add one
molecular size marker every 24 lanes, this will still leave
enough room to load one full 96-PCR plate.
- Run for 100-120 min at 300V. As an alternative, gels can
be run overnight (12-14 hours) at 60 V.
The gel can be reloaded, four times on average. The buffer should
be replaced every two runs.
1 6X loading buffer:
- 10mM Tris-HCI, pH7.5
- 50mM EDTA
- 10% Ficoll 400
- 0.25% Bromophenol blue
- 0.25% Xylene cyanol FF
Experimental conditions presented here should be consider only as
a starting point. It is possible that the procedures need adjustments
to work adequately in a given lab.
Mention of trademark or proprietary products does not constitute a
guarantee or warranty by the authors and does not imply its approval
over other suitable products.