I. AMPLIFY THE GENE (increases/normalizes template concentration for sequencing reaction)

1. PCR the following:

6.0 uL ---10x PCR buffer
3.6 uL ---25mM MgCl2
2.4 uL ---10mM dNTPs
1.2 uL ---10uM primer 1: T7P-M13 (or T7P)
1.2 uL ---10uM primer 2: T7T, aka T7 Term
1.0 uL ---mini prep (pET 15b expression construct) template
0.15 uL ---Taq polymerase
45 uL ---H2O

Through these cycles:
1x (95 C; 60 sec)
35x (95 C; 30 sec); (50 C; 30 sec); (72 C; 40 sec)
1x (72 C; 10 min)

2. check PCR product on 2% agarose gel

II. PURIFY THE SEQUENCING TEMPLATE (removes primers and dNTPs which destroy the sequencing reaction)

Using G-50 w/spin column
1. rinse old spin column under faucet
2. add ~300 uL H2O to spin column, quick spin in microfuge
3. add 600 uL Sephadex G-50 slurry (1g/15mL in ddH20) to spin column
4. microfuge @ 4900 RPM exactly 60 seconds
5. load 20 uL of above PCR product to the center of the top surface of the Sephadex
6. microfuge @ 4900 RPM exactly 60 seconds into fresh 1.5 mL eppendorf tube

Using Sephacryl and Silent Monitor plates
1. load 350 ul Sephacryl S-500 high resolution to each well in Silent Monitor plate.
2. Spin @770x g for 3 minutes using Juan centrifuge into waste strip tubes.
3. Replace strip tubes with new ones to collect samples. Load all of the PCR rxn into the center of the Sephacryl without touching the resin with the pipet tip.
4. Spin @770x g for 8 minutes using Juan centrifuge.

III. RUN THE SEQUENCING REACTION

PCR the following:

5 uL Sephadex/Sephacryl-purified PCR product
1.0 uL 10uM primer 1 (whichever one you used in the above PCR reaction)
4.0 uL Big-Dye Terminator RR Mix

through program "Big-Dye" in the thermal cycler in J579.

IV. PURIFY THE SEQUENCED PRODUCT (removes dye-ddNTPs which destroy the sequencing gel)

Repeat previous spin column purification.

Alternatively, you may EtOH precipitate samples
1. Add 30 ul ddH2O, 60 ul 100% EtOH.
2. Cover micro amp tray with 3M Aluminum Tape making sure the strip tubes are well sealed. Invert tray a few times to mix sample.
3. Leave at room temp for at least 15 minutes. (you can transfer solution to 1.5 ml eppendorf and spin at full speed for in a microcentrifuge 10 minutes instead of steps 4-6 or go to step 4.)
4. Spin 2000 x g for 45 minutes. (Juan centrifuge)
5. Remove supernatant by inverting tray over sink.
6. Place inverted tray with a towel into Juan centrifuge and spin @700 x g (once 700 x g is reached stop centrifuge).

V. DRY/SUBMIT SAMPLE

1. with cap open, allow to air dry--may be placed in 37 C hot block to speed dry.
2. submit to Biochem sequencing facility.