2.
Set up your reaction in PCR tubes: add big dye last
**Terminator Ready Mix should be kept on ice and covered with aluminum foil when not at -20  C.
Terminator Ready Mix (big dye)
Template (your sample)
Double stranded
Single-stranded
8  l ----- this is light sensitive. Thaw on ice, wrap in foil, use 4  l big dye + 4  l buffer (from Jenn, or ddH
2
(1  l)
O)
200-500 ng
50-100 ng
PCR product
Primer
Deionized Water
TOTAL
30-90 ng
3.2 pmol (use T7 in freezer box - 1pmol/  l = 3.2
 l) q.s. (7.8
 l)
20  l
3.
Mix well and spin briefly (or just flick).
4.
Sign up for cycle sequencing (thermocycler by Suzi or in other lab)
5.
Twist lid clockwise til it lifts up, load samples, and close.
6.
Proceed to sequence program - 3 hrs. (overnight):
Rapid ramp to 96  C
96  C for 30 seconds
Ramp (1  C/sec) to 50  C
50  C for 15 seconds
Ramp (1  C/sec) to 60  C
60  C for 4 minutes
Repeat for 25 cycles
Ramp to 4  C and hold until ready to purify.
7.
Add the following to a 1.5ml microfuge tube:
2  ls of 3M NaOAC
50  ls of room temp 95% EtOH
8.
Add reaction and vortex, place on ice 10 minutes (cover with foil) (no longer, or unincorporated dyes will precipitate). Spin 15-30 min.
9.
Wash with 70% EtOH, spin 5 min, dry in incubator (no need to cover)
10.
Resuspend in 10  ls of HI-Di formamide (in red bag at bottom of freezer)
11.
Vortex, heat at 95  2 minutes, quench on ice 2 min.
12.
Take down to Mayo lab (3 rd floor) - BEFORE NOON. Sign up with sample name
(KM1, KM2, etc), Chemistry (BD), CUFS (0985 300 D358), make sure your tubes are labeled.
1.
THE NEXT DAY
Get sequences on line - go to "fetch".
Host = 165.124.234.175
UserID = _3100data
Password = sequence
Directory = users/Evanston
2.
Highlight your data (date sequenced), in binary mode, click on "Get File"

3.
Save to new folder, go to "Conversion Programs" on desktop, "win to Mac", find your new folder, select with one click and hit "Choose".
4.
Open your sequence with Mac Vector. The first 50 bp near beginning and end will be messy.