Pre-siliconize plates and one Eppendorf tube per oligo with Sigmacote®.
- Resuspend (or dry down) oligo to 33 µg/µl (OD260) in H2O.
- Take 8 µl 33 µg/µl oligo + 8 µl formamide and heat at 90°C for 2 minutes.
If you need more than 33 µg of oligo, run more than one lane.
- Make a medium-sized 15% PAGE sequencing gel. Pre-run gel for ~15 minutes, and then flush wells. Run a separate lane with Sequencing Stop Buffer, which contains bromophenol blue (BMB) and xylene cyanol (XC) for sizes. BMB runs approximately 10 bases and XC runs approximately 55 bases.
- Run at 300V for ~2-4 hours.
- Remove top gel plate and cover gel with plastic wrap, then flip and coax gel off of bottom plate. Then cover other side with plastic wrap.
- Place gel on intensifying screen and look from the top with shortwave UV (minimal time). You should see an intense band corresponding to your oligo plus a smear of smaller, incompletely synthesized DNA.
Notes: If you overload the lanes, you will not get good separation and will end up having a lot of contamination in the "intense band" that you see in an overloaded lane.
Use a marker to circle area on one side of plastic wrap.
- Remove from UV, flip gel over and remove plastic wrap from other side. Cut out indicated area of gel, dice up gel fragment and place in siliconized Eppendorf.
- Add 1ml 0.5M NH4Ac/0.01M Mg(Ac)2. Vortex and incubate 6 hours O/N at 37°C.
- Spin down gel pieces and withdraw supernatant. Rinse gel slices with an additional 0.5 ml same buffer and add to other supernatant.
- Dry down in SpeedVac to about 200 µl. Then precipitate DNA with 1 ml 100% ethanol (The salts will probably precipitate but should dissolve during the ethanol washes.).
- Wash pellet (and salts) with 70% ethanol and spin down DNA.
- Resuspend DNA in 200 µl H2O.
- Extract with phenol:chloroform twice.
- Read at OD260 of 5 µl in 100 µl for concentration.
15% Gel (0.5-mm thickness)
- 31.5 g Urea
- 29.5 ml Acrylamide (40%, 19:1)
- 15 ml 5X TBE
- 8 ml H2O
*From S. Y. Roth Dent: Personal notes 1999