UBL Protocol

for

DNA Sequencing ñ ABI 377 Sequencer

Revised May 2002

Part I:Ý Preparation of 10X Tris-Borate-EDTA (TBE) Buffer

Ý1.Ý ÝMeasure out the following ingredients in a beaker:

16.2 g TRIS,

8.4 g ÝÝ Boric Acid,

1.2 g ÝÝ Disodium EDTA

2.Ý Add DI water and fill to150 mL.Ý Use a stir bar to dissolve ingredients completelyóthis may take several minutes.ÝÝÝ

ý This makes enough buffer for ONE run.Ý Up the recipe as needed.

ý TBE cannot be stored for too long (1 month max) or it will precipitate.Ý Do not make too much at a time.

3.Ý Filter the buffer by suction filtration using the vacuum pump and Milipore filters.

Part II:Ý Plate Assembly

Ý4.Ý Clean the plates if necessary.Ý Use Alconox cleaning solution and hot water, then rinse with DI water. Dry with Kimwipes.

Ý5.Ý Place bottom plate (with the notched ends) on the gray cartridge, watermark facing down.Ý Slide the plate all the way down until it stops.

.....

6.Ý Place spacers on the bottom plate, with the "ears" facing each other (inward).

ÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝ ý Use a couple drops of DI water to secure the spacers; Make sure the spacers line up with the edges of the plate.

7.Ý Place top plate on cartridge, with the watermark facing up.Ý

ý Make sure both plates and spacers are lined up with each other.

8.Ý Secure the plate assembly to the cartridge by rotating the black clamps.

9.Ý Attach the sleeve with orange gasket to the plate assembly.Ý Align the hole in the gasket with the hold in the sleeve. Secure by rotating the black clamps.

ÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝ

ý Make sure that the sleeve is attached properly, or the gel will leak when you pour it!

....

Ý10.Ý Attach the syringe to the sleeve.

Part III:Ý Gel Preparation

Ý11.Ý Add 1,000 μL of DI water to 0.1 g APS (Ammonium Persulfate) in a microfuge tube and vortex to mix.Ý Set aside for later.

Ý12.Ý Obtain a 150 mL beaker and add the following:

18 g urea

26 mL DI water

5 mL 10X TBE buffer

Ý13.Ý Place a stir bar in the beaker and stir gently to dissolve the mixture.

ÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝ

Ý14.Ý Add 5 mL of Lone Ranger acrylamide gel solution.Ý Continue stirring for 5 minutes.

ÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝ ý Warning!Ý Actylamide is extremely toxic.Ý Exercise caution and do this in a hood.

Ý15.Ý Connect the vacuum pump to the filter apparatus and turn the pump on.

ÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝ ý Be sure to use the hose attachment that comes with the filter.Ý Do not connect the hose directly to the filter.

Ý16.Ý Pour the gel solution into the filter.Ý The vacuum should draw the solution through the filter into the bottom.

Ý17.Ý Leave the pump on and degas for 10 minutes.

Ý18.Ý Turn off the pump, remove the filter, and put the cap on.

Ý19.Ý Place the gel solution in ice for 5 minutes.

Ý20.Ý Add 250 μL of APS and 35 μL of TEMED.

Ý21.Ý Swirl gently to mix, then pour the gel into the syringe.Ý

Ý22.Ý Immediately start tapping in front of the gel as necessary to prevent bubbles from forming.

Ý23.Ý When the gel goes past the notches of the spacers, remove the syringe and drain the excess back into the container.

Ý24.Ý Insert the flat side of the shark tooth comb and clamp with 3-4 binder clips.

....

Ý25.Ý Remove the plastic sleeve with the orange gasket and seal the bottom of the plates with plastic wrap.

Ý26.Ý Flush the sleeve, gasket, and syringe with DI water and put away for next use.

Ý27.Ý Cap the container with the excess gel solution (acrylamide sublimates!)

Ý28.Ý Allow the gel to polymerize for 2 hours.

Part IV:Ý Instrument Preparation

Ý29.Ý Check the coolant level of the instrument ñ top off if necessary.

Ý30.Ý Check that the gel has polymerized (check the container with the excess gel to see.)

Ý31.Ý Remove the binder clips, comb, and plastic wrap.

Ý32.Ý Take the plate assembly to the sink and wash off any polyacrylamide "gunk" from the bottom and the well with DI water.

Ý33.Ý Dry the plates thoroughly and check that the read region (the area covered by the laser bar) of the plates is clean.Ý

Ý34.Ý Regions of the gel where there are bubbles cannot be used. Take a piece of paper and mark those locations. Overlay the paper with the sample membrane and note the lane numbers that need to be skipped.

.........

Ý35.Ý Attach the lower buffer chamber to the instrument and fill with 1X TBE buffer (dilute the 10X you made earlier 1:10).

ÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝ

Ý36.Ý Attach the plate assembly to the instrument and secure by rotating the black clamps.

ý Be sure to close the laser cover and secure with the black clamps.

Ý37.Ý Turn on the instrument and the Baby Blue Mac that runs it.

Ý38.Ý Start a new sequencing run (FILE > NEW > SEQUENCE RUN) and perform a plate check on the laser read region. If the plates are clean there should be no peaks in the scan window.

ÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝ ý If there are significant peaks, take the plate assembly down and try cleaning them again.

Ý39.Ý Areas of the read region that are dirty cannot be used. Note any additional lanes (if any) that need to be skipped by overlaying the sample membrane to the plate check window on the computer screen.

Ý40.Ý Once the plate and lane checks are complete, cancel the run.

Ý41.Ý Attach the heating unit to the instrument.Ý

ÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝý Do not rest the heating unit on the laser cover ñ it's not designed to carry the weight.

ÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝý Do not mount the heating unit too high, or it will interfere with the upper buffer chamber.Ý You can test-fit the upper buffer chamber now to check.

Ý42.Ý Create a sample sheet for the run (FILE > NEW > SEQUENCING SAMPLE).Ý Do not use the skipped lanes, and be sure to select the correct BigDye matrix.

Ý43.Ý Load the new sample sheet into the sequencing run..ÝÝ

Ý44.Ý Select the correct BigDye matrix and change the run time to 9.0 hours.

You are now ready to load the samples and start the run.Ý If there is a delay, cover the top of the gel (the well) with plastic wrap to prevent the gel from drying out. The picture below shows the completed instrument assembly at this step:

Part V:Ý Starting the Run

Ý45.Ý Pipet the samples into the well loader, using the well numbers and sample IDs that correspond with the sample sheet.

Ý46.Ý Spread 400 μL of ficoll loading solution evenly into the well.

Ý49.Ý Dip the loading membrane into the well loader for 10 seconds.

Ý47.Ý Transfer the membrane to the gel.Ý Align the center of the membrane (there is a notch) with the center of the gel plates (the watermark).ÝÝ

Ý48.Ý Attach the upper buffer chamber (secure with clamps) and fill with 1X TBE.

Ý49.Ý Make sure all connections are correct, then close the door.

Ý50.Ý Start the run, then PAUSE it after 1 minute.

Ý51.Ý Remove the membrane, then flush out the ficoll with a syringe and buffer.

Ý52.Ý Place the cover on the upper buffer chamber and close the door.

Ý53.Ý Resume the run.Ý

Ý54.Ý Open the status window to ensure that all settings are correct.

Part VI:Ý Shutdown

Ý55.Ý Turn off the instrument.

Ý56.Ý Remove the heating unit.

Ý57.Ý Remove the entire plate assembly (including the upper buffer chamber) and take it to the sink.

Ý58.Ý Remove the upper buffer chamber and discard the buffer.

Ý59.Ý Remove the lower buffer chamber from the instrument and discard the buffer.

Ý60.Ý Remove the plates from the cartridge assembly and take it to the table.

Ý61.Ý Separate the plates carefully with a razor blade

ÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝ ý Do not attempt the pry the plates apart with the razor blade ñ the blade will break.

Ý62.Ý Remove the polyacrylamide layer with a paper towel or kimwipe using the "fruit roll-up" method.

Ý63.Ý Wash the plates with Alconox and hot water, then rinse with DI water.

Ý64.Ý Dry the plates with kimwipes, then store them in the drawer.

Ý65.Ý Clean everything else (spacers, buffer chambers, cartridge) with DI water and let them air dry.

Ý66.Ý Remove any buffer or coolant residue from the instrument with DI water and kimwipes.