This is how I was taught to sequence by Wolfe in October of 1993

Primer preparation:
Primers from Denise are good, but require a few steps
• heat at 56°C for 4 hours with lid tightly screwed on
• speed-vac at high speed, in 4 aliquots of ~200µl until dry (~3 hours)
• re suspend one aliquot in 500µl sterile water. Store frozen.

DNA preparation:
DNA to be sequenced should be clean and pure. Concentration can be from 0.4µg/µl to 2.2µg/µl in TE or sterile water.

Materials: takes ~1 hour and 40 minutes to process up to 10 samples
sterile water 2M NaOH + 2mM EDTA
2M ammonium acetate pH 4.6 95% ethanol at -0°C
bucket of dry ice centrifuge at 4°C
70% ethanol at -0°C speed-vac on high heat

In one tube add:
30µl DNA
70µl water
10µl 2M NaOH + 2mM EDTA mix by pipetting
incubate 5 minutes at room temperature
10µl 2M ammonium acetate pH 4.6 mix, then immediately add
375µl ice cold 95% ethanol mix by inverting
incubate *15 minutes on dry ice to precipitate

Centrifuge *15 minutes top speed then aspirate out supernatant
200µl ice cold 70% ethanol to wash
centrifuge 5 minutes top speed then aspirate out supernatant
• speed-vac for *25 minutes until pellet is dry

* Things to do during incubation times
thaw ddNTP’s label 0.65ml tubes with GATC
aliquot ddNTP’s, 2.5µl for each tube, freeze until ready to use

* Things to do during speed-vac
turn water bath to 65°C thaw primers and sequenase buffer

Materials
primers sequenase buffer
sterile water 65°C water bath & bubble rack
1 liter beaker & thermometer ice bucket with ice
centrifuge at 4°C

To template DNA pellet add:
1.5µl primer add right to pellet, resuspend
2µl sequenase buffer mix
6.5µl water mix very well by re-pipetting

Incubate mixture in 65°C water bath for 5 to 7 minutes
• allow to cool slowly to ² 35°C by putting sample in 1 liter beaker of water from 65° bath and placing on bench top. Takes ~90 minutes.

Turn water bath to 37°C and heating block to 95°C

Put ddNTP in 37°C block

Acrylamide solution — For each gel make:
25.2g Urea
7.2ml 10x TBE
6ml Long Ranger acrylamide
to 60ml with Milli-Q: takes some time to go into solution

Clean plates: line sink with mesh to keep from chipping edges of plate
use liquinox, hot water, and scotch-pad to make plates absolutely clean
rinse with DI water, then dry
lay plates flat on Styrofoam racks to keep from chipping edges
siliconize small glass plate in hood, with gloves, by pouring some silane onto center of plate then immediately spreading over entire plate with kimwipe
avoid getting silane on "outside" of plate, or gel sealing tape won’t stick!
allow to dry in hood
wipe both plates with 70% ethanol then 100% acetone

Assemble plates
put spacers on large plate with rubber seal at top of plate
carefully lay small plate on top and align perfectly
clamp at middle of sides
seal bottom and a bit of both sides with gel sealing tape
remove clamps and with one piece of tape seal all of both sides and bottom

Pour plates
put gel on casting stand
filter acrylamide then add 300µl freshly made AMPS and 30µl TEMED
pull acrylamide into 60ml syringe, and slowly squeeze into gel from corner
tap out any bubbles with rubber "cork" on end of pipette
slowly lay gel flat, and insert flat side of teeth until teeth are flush with end of large glass plate
use 3 clamps, one in center then one on each side of top of plate
allow to polymerize for at least 1 hour (if overnight, use Saran-wrap to keep wet)

Before running gel, clean junk at top of gel and remove tape at bottom
pre-run gel at 60W for at least 45 minutes before loading samples
gel should run between 45°C and 50°C

Assemble apparatus
make sure that drain valve on right side is closed (turn clockwise)
remove comb, clean acrylamide from where comb was, and take tape off bottom of gel
place smaller glass plate against apparatus and secure gel against apparatus
pour enough TBE in top chamber to cover top of gel, and pour rest into bottom chamber

Pre-run gel
turn volts and ampere knobs to maximum, limit power to 65 watts
connect leads to power source and gel, then turn on current for at least 45 minutes
temperature should reach 45°C to 50°C

Insert combs so that tips are 2mm to 3mm below top of gel. Never pull combs out again.

Load samples process no more than 4 sets of GATC samples at once
heat samples at 95°C for at least 2 minutes
spin to get entire contents to bottom of tube
turn off power to gel and disconnect negative lead
load 3.3µl per lane, (I load them in this order: GATC)
reconnect leads to power source and turn on current

To read samples close to primer, run for 1hr 40 minutes, or until dark blue dye is 3 cm from bottom of gel

To read sequences far from primer, run for 3 hours and 30 minutes, or until light blue is near bottom of gel

Dry gel
disconnect power and leads from gel apparatus
lay gel flat onto Styrofoam rack, small plate facing up, and remove tape
gently insert prying tool between plates and separate small glass plate from gel.
be careful not to rip gel, especially near bottom. Sides are not so critical
place filter paper onto gel and lift gel from large glass plate. handle carefully
cover gel only (not entire filter paper) with Saran Wrap
place on gel dryer with heat on high for at least 30 minutes

Expose to film at room temperature overnight with intensifying screens

If exposure was satisfactory, dispose radioactive gel in proper waste container