VI. DNA Sequencing

A. CEQ Dye Terminator Cycle Sequencing

1. Example Sample sheet (make like to a compatible excel file)

Gene
	1	2	3	4	5	6	7	8	9	10	11	12
A	1	9	17	25	33	41	49	57	65	73	81	89
B	2	10	18	26	34	42	50	58	66	74	82	90
C	3	11	19	27	35	43	51	59	67	75	83	91
D	4	12	20	28	36	44	52	60	68	76	84	92
E	5	13	21	29	37	45	53	61	69	77	85	93
F	6	14	22	30	38	46	54	62	70	78	86	94
G	7	15	23	31	39	47	55	63	71	79	87	95
H	8	16	24	32	40	48	56	64	72	80	88	96

2. Reaction mix Total Sample: File name:

Chemicals	Stock	Per reaction 10µl	Total volume
DNA termplate		4µl
RNase/DNase free water		1µl
Primer	2µM	1µl
DTCS quick start master mix		4µl
Total		10µl

3. PCR program (55bec30): 3.1 hours in total. Start: _____finish: ______.

1. 95ºC hold
2. Step 1: 96ºC 20 sec
3. Step 2: 96ºC 20 sec, 55ºC 20 sec 60ºC 4 min
4. Step 3: 60ºC for 4 min
5. Step 4: Hold temperature: 4ºC

1. Ethanol Precipitation of Sequuencing reactions

a. Prepare fresh Stop Solution/Glycogen mixture in 1.5ml microtube (The following components are for 2 plates 192 samples. Dispense 67µl in 8-strip tube if necessary):

110µl of 20mg/ml of glycogen

220µl 100mM Na₂-EDTA (Ph 8.0)

220µl 3M Sodium Acetate (pH 5.2)

b. Separate the PCR reaction tubes in 2 racks: 1 rack with forward primer reaction, the other rack with reverse primer reaction.

c. Dispense 2.5µl mix to each PCR tube and mix thoroughly (leave tube open).

d. Spin at 300rpm for 30 seconds on the rack.

e. Add 30µl cold 95% (v/v) ethanol/dH₂O from –20ºC freezer, put lid on and mix thoroughly by inverting 3 times.

6. Immediately centrifuge at 5600rpm for 20 minutes.
7. Discard lids. Invert onto 2 paper towers (fold once).
8. Spin racks upside down at 300rpm for 30 seconds.

i. Add 150µl cold 70% (v/v) ethanol/dH₂O from –20ºC freezer.

1. Immediately centrifuge at 5600rpm for 7 minutes (do not invert).

1. Invert onto 2 paper towers.

12. Spin racks upside down at 300rpm for 30 seconds.
13. Add 150µl cold 70% (v/v) ethanol/dH₂O from –20ºC freezer.
14. Immediately centrifuge at 5600rpm for 7 minutes (do not invert).
15. Invert onto 2 paper towers.
16. Spin plates upside down at 300rpm for 30 seconds.
17. Turn pump power on, Gel pump on, put sample in, turn concentration on, set temperature to low and turn vacuum on (on Close). Dry in speedvac for 1 hour.
18. Turn air button to O slowly then to open, turn concentration off, gel pump off, turn power off, open the speedvac.
19. Resuspend the sample in 40µl Sample Loading Solution (606083).
20. Spin plates at 300rpm for 30 seconds.
21. Mix samples up and down gently using pippet tips and transfer the resuspended samples to the CEQ sample plate (P/N609801, nontransparent).
22. Spin plates at 500rpm for 2 minutes.
23. Dispense 285µl CEQ Separation Buffer (608012, leave at 4ºC) to another plate (transparent).
24. Overlay each of the resuspended samples with one drop of light mineral oil (Sigma Cat#M5904).
25. Load the sample plate into the CEQ and start the desired method.