cDNA phage stock and in vivo excision


Phage stock

  1. Start an overnight of XL1-Blue from a fresh plate in LB (no antibiotics)
  2. Add 100 l XL1-Blue o/n to 3 ml of lambda top agarose at 50°C
  3. Vortex for 10 sec
  4. Quickly pour onto pre-warmed LB plates, tilt plate to spread evenly
  5. Spot 1-3 l (usually use 2 l) of phage stock onto plate using pipettor; can do 4-6 cDNAs on one plate if careful
  6. Incubate o/n at 37°C to get large plaque
  7. Transfer plaque using big end of pasteur pipet to 0.5 ml SM in micro c/f tube
  8. Add one drop of chloroform
  9. Cap well and vortex
  10. Store at 4°C, do not freeze

In vivo excision

  1. Start an overnight of XL1-Blue from a fresh plate in LB (no antibiotics)
  2. Combine 200 l of o/n XL1-Blue, 100 l of phage stock, and 1 l M13K07 helper phage in 50 ml orange cap tube
  3. Incubate tube at 37°C for 15 minutes without shaking
  4. Add 5 ml of RT LB to tube
  5. Incubate 3 hours at 37°C with shaking
  6. Heat tube at 70°C for 20 minutes
  7. Spin tube for 5 min at 6000 rpm, 4°C, fixed angle rotor
  8. Decant s/n into 15 ml orange cap tube--this is your packaged phage particles!
  9. Store at 4°C
  10. To rescue phagemid, combine 20 l of a 10-2 dilution (10 l of packaged phage stock into 990 l of RT LB) and 200 l of o/n XL1-Blue
  11. Incubate at 37°C for 15 minutes without shaking
  12. Plate 10 l onto LB Amp plates
  13. Incubate o/n at 37°C. Colonies on plate contain the pBluescript double stranded phagemid with the cloned DNA insert