Overview and protocol for preparing and running the ABI 377 DNA sequencer
1. Description of Process
The casting and electrophoresis of a DNA sequencing gel.
2. Chemicals involved in the Process
Refer to SOP 2- Preparation of Acrylamide Solution for Sequencing Gels.
3. Potential Hazards
Electrocution
Also, refer to SOP 2- Preparation of Acrylamide Solution for Sequencing Gels.
4. Personal Protective Equipment
Wear a lab coat, enclosed shoes, safety glasses or goggles and nitrile gloves for eye and skin protection.
5. Engineering/ Ventilation Controls
Ensure that the air conditioner next to the ABI 377 sequencer is turned on. The sequencer is susceptible
to overheating.
6. Special Handling Procedures and Storage Requirements
Recap bottles immediately after use.
Store source bottles of flammables in the flammables cupboard and source bottles of corrosives in
the corrosives cupboard
Working stocks of solutions are to be kept in glass bottles and labelled correctly.
7. Spill and Accident Procedures
Skin Exposure: rinse the affected skin with plenty of water while removing contaminated
clothes and shoes. Rinse for at least 15min. Seek medical attention.
Eye exposure: Wash eyes for at least 15min, lifting the upper and lower eyelids.
Seek medical attention immediately.
Small Spills: Notify others in the area of a spill. Do not attempt to clean up
if you feel unsure of your ability to do so or if you perceive the risk to be greater than the normal
laboratory operations. Cover spill with absorbent material (kitty litter) supplied in the Spill Kit.
When absorbent is removed, wash contaminated area.
Large Spill: Notify others in the area of a spill. Turn off ignition sources in
area. Evacuate area and post entrance ways to spill area. Call 4666 in the event of an emergency. Restrict
persons from the spill area until the clean up is complete. Remain in the area in a safe location to
assist with response by emergency services.
8. Decontamination Procedures
Use bicarbonate solution for acids. Use citric acid solution for bases.
9. Waste Disposal Procedures
Disposal of the accumulated waste in the appropriate waste disposal container.
10. Material Safety Data Sheet Location
MSDS folders located in Room 302, Applied Sciences 2
11. Protocol
Prepare the acrylamide gel solution (3.8%) as described in SOP 2- Preparation of Acrylamide Solution
for Sequencing Gels and clean glass plates as described in SOP 138.
De-gas the prepared gel solution for 20 minutes immediately prior to casting.
Polish the plates with 95% ethanol (Never clean plates with 70% Ethanol).
Lay out the casting rack and the lower plate on the casting bench.
Pre-wet the spacers with Milli-Q water and position on either side of the lower glass plate.
Cast the gel using a 50ml syringe to feed acrylamide onto the lower plate, while a second operator
slides the top plate over the lower plate.
Lock the casting frame.
Lift the framed gel and lay it on top of 2 parallel plastic tip boxes.
Check the level of acrylamide at the top of the plate, top up if needed.
Position the plastic comb, teeth facing away from the gel.
Leave the gel to polymerise for 25-30 minutes. While the gel is polymerising, prepare:
1400ml of 1 X TBE buffer and prepare
the sample sheet for the run and
enter the sample sheet into the computer.
Clean the end of the plates with 1xTBE and Kimwipes.
Insert the paper comb at the top of the gel.
Position the lower buffer tank in the sequencer and connect the red electrode.
Position the framed gel in the ABI 377 sequencer and lock into place.
Perform a Plate Check (this takes approx 5 minutes). If the plate check indicates that the plates
are dirty, then carefully re-clean the area of plate that the lazer identified as dirty. After re-cleaning,
re-perform the plate check.
Seal the shoulders of the comb area in the plate with 2% agarose.
Attach the upper buffer tank to the framed gel and connect the black electrode.
Fill both the upper and lower buffer tanks with 1xTBE buffer.
Pre-run the gel for 20min.
While the gel is pre-running, denature the prepared sequencing samples by heating the samples at
90oC for 2min.
Place denatured samples on ice.
Using the large needled syringe (stored on top of the sequencer) run a blast of 1xTBE along the
top the comb to rinse out wells.
Load 0.5-1ul of sample, every odd lane (1,3,5,7,9 etc).
Start the sequencing run.
Run for two mins (no longer!)
Pause the run.
Using the small needled syringe (stored at the top of the DNA sequencer) individually wash out the evenly
numbered wells.
Load the evenly numbered lanes with the remaining samples. Note: you may have to re-clean wells with the syringe if loading becomes difficult. This will remove
bubbles and the built up urea.
Re-start the run.
Run for 8 minutes.
Pause the run.
Remove the comb.
Re-start the run Note: If 2 sequencing gels are being run a day, the morning run only needs to run for 8 hours.