BAC end sequencing using Qiagen R.E.A.L. Prep Kit (cat#26171)

If doing less than 24 samples, do in tubes (if doing more than that, process in the deep well block).

If using culture tubes, for each sample, fill with 1.5 ml of 1X LB containing appropriate antibiotic, inoculate and incubate at 37^oC for 20-21 hours on orbital shaker. Centrifuge for 10 minutes at 2500, pour off supernatant & tap on paper towel.

If using deep well plate, for each sample, fill with 1.5ml/well of 1X LB containing appropriate antibiotic. Inoculate 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, pour off supernatant & tap on paper towel.

Resuspend pellet in 0.3 ml Buffer R1, vortex. Pellet should be completely resuspended with no cell clumps.

Add 0.3 ml Buffer R2, invert gently 10 times (if doing in deep well block, put compression pad on top of plate & put into compression device, centrifuge up to 2000 RPM then stop, just to get liquid off of the seal) and incubate at room temperature for 5 minutes (do not vortex at this step).

Add 0.3ml Buffer R3, invert gently 10 times (if doing in deep well block, put compression pad on top of plate & put into compression device, centrifuge up to 2000 RPM then stop, just to get liquid off of the seal), wait 10 minutes, spin at maximum speed for 1 minute.

Place new square-well block in the base of QIAvac manifold, reassemble the manifold, and place a QIAfilter plate on the top. Transfer lysates to wells of QIAfilter plate and apply vacuum until lysates are completely transferred to block in the QIAvac base.

Take square-well block with lysates and add 0.63 ml of room-temperature isopropanol, 0.2 ul of glycogen (20mg/ml), tape block, invert 3 times. Put at -20C for 1 hour.

Transfer to tubes and centrifuge for 30 minutes at 6000, invert and tap firmly onto paper towels to remove supernatant.

Add 0.5 ml of 70% EtOH, centrifuge at 6000 for 15 minutes, invert and tap firmly onto paper towels to remove supernatant. Repeat this step.

Spin down and pipette off any additional EtOH. Resuspend pellet in 10 ul H2O. Let sit for at least 10-15 minutes, preferably overnight.

Denature DNA prior to adding sequencing pre mix. (96 C for 5 minutes > 4 C).

Use sequencing pre-mix:
10 ul DNA
2 ul 50 pMol M13 R or M13F primer (10uM)
8 ul mix

PCR 100X {95 C (20s) > 50 C(15s) > 60 C (1 min)}> 4 C

Transfer samples to 1.5ml tubes.

Make master mix:
7ul Ammonium acetate (7.5 M)
75ul EtOH

Add 82 ul of the mix to each sample, spin for 20 minutes at maxiumum speed. Pour off supernatant, wash pellet with 500ul 70% EtOH, spin for 3 minutes at maximum speed.

Pour off supernatant, spin down & pipette off any remaining supernatant. Let dry 5-10 minutes or until completely dry. Put in freezer, fill out paperwork to submit samples to Sequencing Facility.