Cosmid / Low Copy Plasmid Prep¶
Contributed by Ian Chin-Sang, Queens University, ON, Canada
Prep for cosmids or low copy number plasmids.
Requirements¶
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
Method¶
- 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.