SEQUENCING

A. Subclone library construction

Day 1:
1. Inoculate 10 ml of overnight glycerol culture of a BAC clone in 250 ml LB with 25 mg/ml chloramphenicol and shake at 300 rpm, 37 C for 18 hours.

Day 2:
2. Spin down cells at 5,700 rpm for 15 min at 4 C in the Beckman JLA-10.500 rotor.
3. Resuspend cells in 15 ml of buffer P1 with 100 mg/ml RNase A (add RNase A solution before use).
4. Add 15 ml of 0.2N NaOH and 1% SDS, mix by inverting 2 times gently, and keep at room temperature for 4 min.
5. Add 15 ml of pre-chilled 4 C buffer P3, mix by transferring solution to the centrifuge tubes, and keep on ice for 10 min.
6. Spin down cell debris at 12,000 rpm for 30 min at 4 C in the Beckman JA-14 rotor.
7. Transfer cell lysate to a set of clean tubes and spin down cell debris twice at 12,000 rpm for 15 min at 4 C until no debris is present in the supernatant.
8. Transfer the supernatant to a clean tube and mix with 28 ml of isopropanol by rotating and inverting tubes carefully.
9. Spin down the precipitate at 10,000 rpm in a Beckman JA-14 rotor for 30 min at 4 C. Carefully decant the supernatant.
10. Wash pellets with 5 ml of 70% ethanol and spin down the pellet at 10,000 rpm in the Beckman JA-14 rotor for 15 min at 4 C.
11. Place the tube upside down on a paper towel and allow to air-dry for 3 min.
12. Redissolve the DNA in 9.5 ml of buffer EX by gentle shaking.
13. Add 200 ml of ATP-dependent exonuclease and 300 ml 100 mM ATP solution to the dissolved DNA, mix gently, and incubate in a 37 C water bath for 60 min. (Resuspend the one vial of exonuclease in 225 ml of Exonuclease Solvent before use)
14. Equilibrate a Qiagen-tip 500 by applying 10 ml buffer QBT, and allow the column to empty by gravity flow.
15. Add 10 ml of buffer QS to the DNA sample, apply the whole sample to the Qiagen-tip, and allow it to enter the resin by gravity flow.
16. Wash the Qiagen tip with 2x 30 ml buffer QC, and elute DNA twice with 7.5 ml buffer QF each time (buffer QF works better if prewarmed to 65 C).
17. Precipitate DNA by adding 10.5 ml of isopropanol, mix and centrifuge immediately at 11,000 rpm in the Beckman JA-17 rotor for 30 min at 4 C. Carefully decant the supernatant.
18. Wash DNA pellet with 5 ml of 70% ethanol and spin down the pellet at 11,000 rpm in the Beckman JA-17 rotor for 15 min at 4 C. Carefully decant the supernatant without disturbing the pellet.
19. Air-dry the pellet for 10 min and dissolve the DNA in 750 ml of filtered TE buffer.

Day 3:
20. Run some DNA on a gel to estimate DNA concentration. Use Millipore Centricon to reduce volume to ~100 ml, and add TE to adjust DNA to 30 ng/ml.
21. Shear 80 ml of DNA with the HydroShear (GeneMachine) using code 12.
22. Repair the ends of DNA by adding the followings and keep the reaction at room temperature for 40 min.

10 ml Klenow buffer
10 ml dNTP (2 mM)
4.5 ml T4 DNA polymerase (1 U/ml)
4.2 ml Klenow enzyme (5 U/ml)

23. Add 20 ml gel loading dye, incubate at 68 C for 15 min, and run on an agarose gel.
24. Stain the gel with ethidium bromide, and cut out the fragments between 3.5-4 kb.
25. Recover DNA fragments using the QIAquick Gel Extraction Kit (Qiagen). Normally you will recover 48 ml of eluted DNA
26. Precipitate DNA with 2 ng/ml glycogen and 0.35 M NaOAc pH 5.2 and 2.5 volume of ethanol, keep on ice for 10 min.
27. Spin down DNA, wash pellet once with 70% ethanol, and store at -20 C.

Day 4:
28. Dissolve DNA in 15 ml TE
29. Set up ligation as followed, and Keep at room temperature for 1.5 hours

9.75 ml of gel purified DNA
1 ml of 10 ng pUC18 SmaI/BAP vector (Invitrogen)
0.75 ml of 10 mM ATP (Fast-Link)
1.5 ml of 10x ligation buffer (Fast-Link)
2 ml of DNA ligase (Fast-Link)

30. Mix 20 ml of electro-competent cells (Invitrogen) with 1 ml of ligated DNA on ice and transfer into a pre-chilled micro-electroporation chamber.
31. The electroporation is performed using the Cell-Porator (Life Technology) at 410 V with 330 mF capacitance and 4,000 ohms resistance on the voltage booster.
32. Incubate electroporated cells in 2 ml of SOC medium (Invitrogen) at 37 C for 1 hour.
33. Plate out 150 ml of rescued cells on a LB plate with 60 mg/ml X-gal, 0.1 mM IPTG, and 200 mg/ml Ampicilin, and incubate at 37 C for 18 hours. Store the rest of the transformed cells in 10% glycerol at -80 C.

Day 5:
34. Pick colonies into 96-well plates using Q-pix. The cells are picked into LB plus 10% glycerol.
35. Incubate at 37 C for 18 hours and store at -80 C.

B. preparing sequencing templates using rolling circle amplification

1. Thaw denaturing buffer and TempliPhi premix (Amersham, Part No. 25-6400-01) on ice (start thawing at least 3 hours before setting the reaction).
2. Add 2.5 ml of denaturing buffer in each well of 96 well PCR plate.
3. Add 1 ml of cells (taken directly from the glycerol stock) in each well.
4. Spin down and mix the cells and buffer in a quick spin using the table-top centrifuge
5. Denature at 95 C for 3 min
6. Keep on ice for 5 min
7. Add 2.5 ml of premix, spin down and keep at 30 C for 18 hours.
8. Denature at 95 C for 5 min, and store at -20 C until future use.

C. BigDye terminator sequencing

1. Preparing cocktails for sequencing

For each reaction
BDT 0.5 ml
BDT buffer (5x) 1.25 ml
ddH2O 3.25 ml
Primer 2 ml
Primers: 2.7 mM M13.Fwd, 1.6 mM M13.Rev
BDT V3 (Applied Biosystem Part No. 4390246)

2. Dispense 7 ml of cocktail in each well. Add 2 ml of template DNA
3. Sequence reaction setting

96 C 45 sec
96 C 10 sec
50 C 5 sec
60 C 4 min, repeat 96-50-60 25 cycles

4. Add 40 ml of 75% isopropanol, keep at room temperature for 20 min
5. Centrifuge at 4000x g for 40 min
6. Decant supernatant and place on filter paper.
7. Quick spin (~500x g) the plates in the inverted position on a piece of filter paper.
8. Air-dry the plates for 30 min at room temperature.
9. Store at -20 C