BAC purification for injection

This is a two step procedure, utilizing a Qiagen Large-Construct Kit for the initial purification, followed by a simple chromatographic separation.

Qiagen Prep of BAC DNA

Modified from Biotechniques 27:72-74 (1999)
  1. Seed 250 ml LB (containing appropriate antibiotic) with approximately 0.5 ml of an over/day culture (containing appropriate antibiotic); grow overnight.
  2. Spin 15 min at 5500xg to pellet cells; decant supernatant
  3. Resuspend cell pellet in 50 ml P1 Qiagen buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA)
  4. Add 50 ml P2 buffer (200 mM NaOH, 1% SDS); invert slowly 4-6 times; incubate 5 min at RT
  5. Add 50 ml pre-chilled P3 buffer; invert slowly 4-6 times; incubate on ice 30 min
  6. Spin 60 min at 15000xg at 4°C; filter supernatant through Whatman filter paper
  7. Equilibrate tip-500 column with 10 ml QBT buffer (750 mM NaCl, 50mM MOPS pH 7.0, 15% isopropanol, 0.15% Triton X-100)
  8. Apply filtered DNA solution to the column
  9. Wash column twice with 30 ml QC buffer (1.0 M NaCl, 50 mM MOPS pH 7.0, 15% isopropanol)
  10. Elute DNA at 65°C with 5x3ml aliquots of QF equilibrated to 65°C
  11. Add 10.5 ml RT isopropanol to the DNA and mix. Spin 60 min at 15000xg at 4°C; discard supernatant
  12. Wash pellet with 5 ml RT 70% ethanol and spin 15 min at 15000xg at 4°C; remove supernatant; air dry inverted on paper towel for 15 min
  13. Add 60 µl of TE buffer to the bottom of the tube; let stand 30 min at RT
  14. Remove the DNA solution and add 40 µl more of TE to bottom of the tube; let stand 15 min; spin briefly to collect liquid and combine the solutions for total volume of 100 µl

Sepharose Purification of BAC DNA

Modified from Nat Biotech 15:859-864 (1997)
  1. Use pressurized air to blow the cotton plug to the tip of a 5 ml plastic pipette and clamp the pipette on a stand. Shake CL4b sepharose well and gradually add into the plastic pipette until it is packed almost to top leaving about 1 ml space. Do not let the column dry at any point in preparation of the column or during the chromatography.
  2. Use a 10 ml syringe to make a reservoir on top of the column (wrapping parafilm around syringe/pipet junction to prevent leaking). Wash the column three times with 10 ml of the injection buffer (autoclave-sterilized 10 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 100 mM NaCl). This takes 2-3 hours.
  3. Add 5 µl of any Bromophenol Blue-containing DNA loading buffer to the 0.1 ml of Qiagen-prepped BAC DNA. When the buffer has receded to just above the resin bed, remove the reservoir and add the DNA to the column. Wait until DNA+dye just goes into the column, then add 0.5 ml injection buffer to the top of the column.
  4. Reattach the syringe-reservoir with parafilm. Add 10 ml injection buffer. Start collecting 0.5 ml fractions with a 24 well plate (about 12 fractions for the dye to reach the bottom of the column). Circular BAC elutes early, about in fractions 4-7, followed by linear BAC and genomic DNA (about fractions 6-9).
  5. Run 50ul of each fraction on a low percentage agarose gel to identify the early BAC fractions. Typically, the BAC concentration will be 5 to 20 µg/ml. Purified BAC DNA is stable for weeks at 4°C.