Cosmid Prep

Cosmid Prep / Low Copy Plasmid Prep

Reagents & Buffers:

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

 

Procedure:

  1. 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
  2. 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