Cosmid prep or scaled up mini prep

  1. Grow 3-4 tubes of each cosmid in 5 ml media overnight

  2. Harvest a total of 4 ml of each tube separately in 2 ml centrifuge tubes

  3. Resuspend completely in 200 l Solution I*

  4. Add 400 l Solution II* and invert until solution clears

  5. Add 300 l Solution III* and invert gently

  6. Hold on ice for 10 min

  7. Spin 10 min at 4°C and transfer s/n to new tube

  8. Phenol/chloro/IAA one time with equal volume

  9. Spin 1 min at 4°C and transfer upper layer to new tube

  10. Ppt with 0.8 vol isopropanol

  11. Spin 20 min at 4°C

  12. Remove s/n and allow pellet to dry

  13. Resuspend in ~ 50 l of TE + RNase

  14. Cosmids => Digest 5-10 l with either EcoRI or Hind III

    *refer to Alkaline Lysis Solution I, II, or III recipes in card box