BAC End Sequencing Protocol from Mini-preps
developed by ATGC members: Gene Buehler, Ji-dong Feng, Ken Dewar, Yu-Ping Lu

A. Mini-prep Isolation of BAC DNA [adapted from Sheng et al (1995) NAR 23:1990-1996]

1. Incubate single BAC colonies into 3-5 ml of LB medium supplemented with antibiotic (chloramphenicol at 12.5 ug/ml or kanamycin at 25 ug/ml), incubate at 200-250 rpm overnight at 37 C.
2. Collect 3 ml overnight culture in single microfuge tubes by two rounds of centrifugation, spinning at top speed for 10 seconds in a bench top microfuge. Do not centrifuge too long, since longer spins result in pellets that are harder to resuspend.
3. Resuspend each pellet in 100 ul of Solution I (50mM glucose, 25mM Tris-Cl pH8.0, 10mM EDTA pH8.0)
4. Add 200 ul of freshly prepared Solution II (0.2N NaOH, 1% SDS), mix by inversion several times.
5. Add 150 ul of ice-cold Solution III (100ml stock made from 60 ml 5M KOAc, 11.5 ml Glacial Acetic acid, and 28.5 ml H2O), incubate on ice for 10 minutes, occassionally mixing by inversion.
6. Centrifuge at top speed for 6 minutes, then carefully transfer the supernatants to new tubes.
7. Add 1 ml 100% ethanol, mix by inversion, and centrifuge at top speed for 10 minutes. Wash each pellet with 500 ul 70% ethanol, then air dry the samples.
8. Dissolve each DNA sample in 30 ul TE (pH 8.0).

B. Digestion and Purification of BAC DNA

9. Digest each DNA sample with 10-15 U EcoRV in a 50 ul reaction volume for 3 hours to overnight at 37 C.
10. Add 100 ul of 6M NaI and 15 ul glassmilk. Incubate on ice for 5-10 minutes.
11. Centrifuge at top speed for 15 seconds, pour off and discard the supernatants.
12. Add 1 ml of wash buffer (100 mM NaCl, 1 mM EDTA, 10 mM Tris-HCl pH 7.5, 50% ethanol), vortex to resuspend the pellets.
13. Centrifuge at top speed for 20 seconds, pour off and discard the wash buffer. Centrifuge briefly and discard residual wash buffer.
14. Resuspend each pellet in 25 ul H2O, then incubate 5 minutes at 50C.
15. Centrifuge 30 seconds at top speed, then transfer digested DNA samples to new tubes.

C. BAC End Sequencing

16. Use 10 ul of the digested DNA sample for each sequencing reaction (ABI PRISMTM Dye Terminator Cycle Sequencing Ready Reaction Kit). Add 3.2 pmol of primer and 8 ul of sequencing kit stock for each 20 ul reaction.
17. Begin thermal cycling (Peltier Thermal Cycler, MJ Research Inc.): 96 C for 1 minute, followed by 30 cycles of 96C for 20 seconds, 50 C for 5 seconds and 60 C for 4 minutes. Hold at 4 C.
18. Transfer each 20 ul reaction to a 1.5 ml microfuge tube, and add 2 ul 3 M NaoAc pH5.2 and 50 ul 100% ethanol, Mix by vortexing and incubate on ice for 10 minutes.
19. Centrifuge at top speed for 20 minutes, then carefully aspirate all of the liquid from the pellets.
20. Wash each pellet once with 250-300 ul 70% ethanol, centrifuge for 5 minutes, carefully aspirate the ethanol and air dry the pellets.
21. Resuspend each pellet in 2.5 ul of loading buffer (5:1 ratio of formamide:25 mM EDTA/50 mg/ml dextran blue).
22. Denature the samples for 3 minutes at 94 C, centrifuge briefly, then incubate on ice until ready to load.
23. Load 2 ul sample/well on ABI Prism 377.

Notes:
1. The quantity of BAC DNA isolated from 3 ml of overnight culture is sufficient for two sequencing reactions. BAC DNA from 5 ml of overnight culture results in stronger sequencing signals.
2. If EcoRV digestion produces truncated sequences (due to an EcoRV site close to the end of the BAC clone), another restriction enzyme not present in the BAC MCS can be substituted. Alternatively, the BAC DNA can be mechanically sheared or fragmented by acid hydrolysis.
3. Glassmilk can be can also be prepared following reference, or is commercially available (GENECLEAN III KIT, BIO101, Cat #1001-600).
4. Of the various primers tested, M13(-20) 5'-GTAAAACGACGGCCAGT-3' (on the T7 side) and M13reverse 5'-AGCGGATAACAATTTCACACAGG-3' (on the Sp6 side) give consistently good results.
5. Glycogen can be added (to a final concentration of 1 ug/ml) as an inert carrier of DNA when precipitating very low quantities of DNA.