BAC isolation protocol for fluorescent fingerprinting

1. Fill deep well plate with 1.5ml/well of 1X LB containing 12.5ug/ml chloramphenicol. Innoculate plate and cover using Qiagen Airpore tape sheets (Cat# 19571). Incubate at 37^oC for 20-21 hours on an orbital shaker. After 20-21 hours, centrifuge at 3000 RPM, 10 minutes at 4^oC.

Use v-bottom plates, pellets compact easier and arenŐt as easy to disturb when transferring supernatant to the new plate.

2. When done, remove from centrifuge, dump supernatant. Let drain onto paper towels, then tap firmly onto paper towels to get as much supernatant out as possible. If you are not running the plate right away, cover with plastic wrap and put in -20^oC after supernatant dumped. Easier to resuspend pellets if plates at -20^oC at least overnight. Resuspend pellet by adding 110 ul of Solution 1 to each well and vortex until pellet fully resuspended

Solution 1: 50 mM TrisHCL pH 8.0, 10 mM EDTA pH 8.0, 100 ug/ml RNase

3. Important that the next step take no more than 5 minutes or you will start to denature BAC DNA. Start timer when you add 110 ul of Solution 2 to each well. Quickly rotate at an 80^o angle completely around 20 times, don't shake, don't invert

Solution 2: 200mM NaOH, 1% SDS
**Solution 2 only good for 1 week, preferrably make fresh**

**if doing 2 plates, when 1st is at 3 minutes, add Sol II to second plate
spin down after 2nd done & keep in centrifuge 10 minutes**

4. When 5 minutes is up, add 110 ul of Solution 3 to each well. Put seal tape on, compression pad on top, put into compression device, make sure compression pad is centered over plate, tighten screws. Rotate completely around 15 times, donŐt shake. Centrifuge just to get liquid etc. off lid, then keep at 4^oC for 10 minutes

Solution 3: 3M Potassium acetate, pH 5.5

5. Centrifuge at 6000 rpm in SIgma for 15 minutes at 4^oC. Pellets in rows 1-6 will go against left side of the well and pellets in rows 7-12 will go to the right side of the well (clearly seen if hold up to a light).

6. Tip plate onto one corner, so pipette tip is furthest away from pellet, then slowly remove 200 ul of the supernatant and transfer it to a clean 96 well plate, try not to get any of the white precipitate,

7. Add 400 ul 100% ethyl alcohol to each well, replace cover, SEAL TIGHTLY. DONŐT FLIP OVER, quickly rotate at an angle completely around 20 times. Vortex one time each (left, middle, right parts of the plate), at speed 5.5. Keep at 4^oC for at least an hour (I usually left overnight), then spin 6000 for 10 minutes at 4^oC.

8. Remove cover, dump supernatant, drain, tap & get as much fluid out as possible.

9. Add 200 ul of 70% chilled ethyl alcohol to each well. SEAL TIGHTLY, swirl to mix. Don't invert. Centrifuge at speed 6000 for 3 minutes at 4^oC. Remove cover, dump supernatant, drain, tap gently, get as much of EtOH out as possible.

10. REPEAT 70% EtOH wash and spin. Remove cover, dump supernatant, drain, tap gently, get as much of EtOH out as possible.

11. Then let dry in the dryer for 5 minutes

12. Add 60 ul of H₂O to each well, SEAL TIGHTLY. Cover & let sit on bench for 30 minutes, then store at 4^oC if using in day or two. Store at -20^oC if not using right away (probably usable for 3 months if stored at -80^oC)

CK 1/04