C.B.S. Scientific DH Series - Manual (Page 17)

C.B.S. œ Scientific
17
Dedicated Height Sequencer
4. Verify that the upper buffer reservoir drain is secure. For the single 20cm wide sequencer,
fill the upper buffer reservoir with approximately 350ml of electrophoresis buffer. Make sure
no buffer is leaking from the upper buffer reservoir. Fill the lower reservoir with approximately
350ml of electrophoresis buffer. For the dual 20cm wide sequencer, fill the upper reservoir
375ml and the lower with 350ml. For the single 33cm wide sequencer, fill the upper buffer
reservoir with approximately 550ml of electrophoresis buffer. Make sure no buffer is leaking
from the upper buffer reservoir. Fill the lower reservoir with approximately 450ml of
electrophoresis buffer. For the dual 33cm wide sequencer, fill the upper reservoir 450ml
and the lower with 450ml. Be sure no bubbles obstruct buffer contact with the lower edge of
the gel. Note: Do NOT overfill buffer reservoirs or allow buffer to make contact with the
banana jacks.
WARNING: No buffer should leak through or around the silicone gasket or down the side of
the gel assembly. Leakage may allow the upper reservoir to drain dry or cause
arcing and damage to the apparatus.
5. Close the reservoir safety covers to the upper and lower reservoirs. Connect the female end
of the black power cord into the safety cover of the upper reservoir. Connect the female end
of the red power cord into the safety cover of the lower reservoir.
6. Be sure to attach Temperature Strip (cat. # EGT-100) to upper or middle of glass plate, to
monitor the temperature of the gel.
7. Connect the DC power leads to the power supply with the proper polarity. Make sure the
black leads are connected to the black cathode (-) and the red leads are connected to the red
anode (+).
8. If using a rectangular comb, rinse sample wells with upper reservoir buffer. Before loading
samples, pre-electrophorese the gel for 30 to 60 minutes (See Table 5.1 for recommended DC
power settings). Best results are achieved with a gel surface temperature of 45
°-50°C.
WARNING: Excessive power will cause the gel to overheat and crack the glass plates.
4.2 Loading
Samples
1. Prepare the samples by heating in an appropriate loading buffer 95
°C to 100°C for 3-5
minutes, vortexing, and then chilling immediately on ice.
2. At the end of the pre-electrophoresis period, TURN OFF THE POWER SUPPLY AND
DISCONNECT BOTH DC POWER CORDS FROM THE POWER SUPPLY. Remove power
cords from both reservoirs. Open upper reservoir safety cover.
3. Immediately prior to loading the samples, rinse the wells of the gel thoroughly with
electrophoresis buffer. Use a sequencing loading tip or a syringe with a bent needle to wash
away urea that has diffused into the wells. Load samples in a loading pattern of your
preference. For sharktooth combs load 2-3ul of sample. (For rectangular tooth combs see
comb volumes, Table 5.3).