BAC/FOSMID Mini-Prep protocol (entire protocol at room temp)

Inoculate 1.5 ml of 1X LB with 12.5 mg chloramphenicol (in culture tubes or deep well plates) and grow overnight at 37^oC.

Transfer samples to 1.5ml tubes and spin 1.5ml of culture for 30 seconds at full speed in microfuge, discard supernatant. Can freeze the pellet at this stage and process later.

If incubated in deep well plate, spin at 6000 RPM for 30 seconds in centrifuge, discard supernatant. Can freeze the pellet at this stage and process the samples later.

After adding 100ul Solution 1 to either a 1.5ml tube or sample in a deep well plate, pipette up and down to mix, vortex briefly to make sure no clumps of cells remain.

****After adding Solution 2, you need to add Solution 3 within 4-5 minutes.****

Start timer when adding 200ul of Solution 2 to first sample, cap and gently rotate (not invert quickly) tubes 7-9 times. If doing in plates, don't invert, rotate 10 times completely around quickly, holding plate at about a 60 degree angle. Incubate 4-5 minutes from time Solution 2 was added to first sample.

Add 150ul of Solution 3, cap and gently rotate (again, not inverting) 7-9 times. Spin tubes for 6 minutes at full speed in the microfuge or if doing in deep well plate, don't invert, quickly rotate 10 times completely around, holding plate at about a 60 degree angle, then spin at 6000 RPM for 6 minutes.

Transfer supernatant to fresh tube (or fresh plate) using large orifice pipette tip or tip with end cut off. Add 1 ml 100% EtOH, if in tube, mix by inverting, and spin 10 minutes at full speed in microfuge. If doing in plate, don't invert, instead quickly rotate 10 times completely around, holding plate at about a 60 degree angle, then spin 10 minutes at 6000 RPM.

Pour off supernatant, add 500ul of 70% EtOH to rinse pellet. Pour off & drain tube (or plate) upside down on clean paper towel. Dry completely. Resuspend pellet in 20ul H₂O and place at 4C. Use DNA within a couple days.

Solution 1 is 50 mM glucose, 25 mM Tris-Cl, pH 8.0, 10 mM EDTA, pH 8.0

Solution 2 is 0.2N NaOH, 1% SDS

Solution 3 is made by adding a glacial acetic acid to a solution of 3M potassium acetate to achieve a pH of 4.8. This is accomplished by adding a minimal amount of water to potassium acetate and then adding acetic acid until potassium acetate is dissolved and the pH has reached 4.8

Hint: Can use R1, R2, R3 from Qiagen R.E.A.L. Prep Kit (cat#26171) in place of Solutions 1, 2, 3 or P1, P2, N3 from other Qiagen kits.