ProCipitate is a fast method to produce protein and enzyme free BAC/PAC preparations from small-scale (10ml) preparations. No CsCl ultracentrifugation is necessary however the host E.coli genome is not removed. The DNA can be directly sequenced with Big Dye Terminators.
[ProCipitate suppliers are Ligochem (URL http://www.ligochem.com)]

Set up overnight cultures from single colonies or directly from the -70°C glycerol stock:
BAC clone - 2 x 10ml (2xTY + 10µl of chloramphenicol [25mg/ml]).
PAC clone - 2 x 10ml (LB + 10µl kanamycin [25 mg/ml]).
Incubate overnight at 37^oC in a shaking incubator.

• Split the first culture into 4 x 2ml and spin in micro centrifuge for 10 mins. at 13,000 rpm.
  

• Discard the supernatent and add another 2ml from the second culture to each of the tubes, spin as before.

• After spinning, what remains is a 4 tubes, each with a pellet from 4ml of culture.

• To each tube add 300 µl of GTE* and 10 µl of RNAse (stock concentration of 10 mg/ml).
  

• Vortex gently for 5 min until resuspended.
  

• Add 400 µl 1% SDS/0.2M NaOH** to the suspension, invert continuously for 5 min until well mixed.
  

• Add 200 µl of 3M KAc to each tube, then 100 µl of ProCipitate. Ensure thorough mixing of the ProCipitate container during its addition as it quickly settles out.
  

• Invert as before for 5 min. until well mixed.
  

• Spin for 15 min.in micro centrifuge at 13,000rpm.

• Remove supernatant and place in a fresh tube, taking care not to disturb the residual pellet which will be soft and fluffy. (If a small quantity of pellet is carried over it will be necessary to respin the sample)

• To the supernatant add 2 volumes of absolute ethanol and spin for 15 minutes at 13,000 rpm.

• Remove ethanol carefully and wash pellet with 70 % ethanol. Then spin for 10 minutes at 13,000 rpm.

• Remove the ethanol wash and dry the pellet in a vacuum centrifuge for 20 minutes.
  

• When completely dry, add 50 µl of 1x TE to each tube, and whirlimix gently then leave overnight at 4°C for the DNA to re-suspend.
  

• The DNA is now ready to be quantified by spectrophotometry: Measure the OD₂₆₀ of a 1 in 100 dilution (5µl DNA + 495 dH₂O).
  An OD of 0.5 = 1.0mg/ml = 1.0µg/µl (multiply OD x2). Normal yields are about 200µg (1µg/µl)

*10ml GTE = 0.5ml 20% Glucose, 0.25ml 1M Tris/HCL pH 7.5, 0.2ml 0.5M EDTA pH 8.0, 9.05 ml dH₂O

**10ml 1% SDS/0.2M NaOH = 0.5ml 20% SDS, 4ml 0.5 M NaOH, 5.5ml dH₂O.

Version 1.1;1 1/12/98