Cosmid / Low Copy Plasmid Prep

Contributed by Ian Chin-Sang, Queens University, ON, Canada

Prep for cosmids or low copy number plasmids.


LB: 10 g tryptone, 5 g yeast extract, 10 g NaCl per liter (pH to 7.0) Resuspension buffer: 50 mM Tris, 10 mM EDTA (pH 8.0) Lysis buffer: 200 mM NaOH, 1% SDS 4 M KOAc: pH to 5.3 with glacial acetic acid Qiagen tip-100 QBT buffer:0.75 M NaCl, 50 mM MOPS, 15% isopropanol, 0.15% TritonX-100 (pH 7.0) QC buffer:1.00 M NaCl, 50 mM MOPS, 15% isopropanol (pH 7.0) QF buffer:1.25 M NaCl, 50 mM Tris, 15% isopropanol (pH 8.5) Isopropanol 70% ethanol


  • Grow up cells from which to purify DNA
  • Innoculate 100 mls LB + appropriate antiobiotic with 1 ml saturated culture
  • Grow overnight or to saturation
  • Spin down cells 7’ @ 5000K (SLA-1500 rotor)
  • Resuspend cells in 5 mls resuspension buffer - make sure cells are thoroughly resuspended
  • Transfer cell suspension to 50 ml conical tube
  • Lyse cells to release cosmid DNA
  • Add 5 mls fresh lysis solution and invert gently twice to mix
  • Incubate on ice for 5 min
  • Add 5 mls 4 M KOAc and invert gently 2-3 times to mix
  • Incubate on ice for 10 min
  • Prepare an empty 50 ml syringe barrel by pluging a KimWipe into the bottom
  • Place syringe over clean 50 ml conical tube
  • Dump cell lysate into syringe and allow clarified solution to drain into 50 ml conical tube while the precipitate will be captured atop the KimWipe
  • Add RNase to 50 ug/ml and let sit at room temperature while equilbrating Qiagen column

This method is based, with permission, on an original protocol available here.