Difference between revisions of "PhiX:DsDNA isolation"

Latest revision as of 16:37, 22 February 2010

Developed after Godson and Vapnek, 1973.

Step One: phage amplification

• prepare ~20ml of high titre phage (I have tried this from chemostat where pfu = 10⁹/ml)

Step Two: stalled in vivo replication

• prepare 100ml LB + 200ul 1M CaCl₂ in a 250ml conical flask (~ 2mM CaCl₂)
• add 2ml overnight C122 culture to each
• shake at 37^oC and follow until 0.3 < OD_600nm < 0.4 (~1.5 hours)
• add ~20ml phage - start timer
• return to shaker for 9 minutes (within the time of a single burst)
• remove from shaker and quickly add 88.2ul 34mg/ml chloramphenicol (~ 30ug/ml)
• shake for 3 hours at 37^oC
• aliquot culture into four 50ml centrifuge tubes
• centrifuge @ 5000g for 20 minutes (4^oC to avoid overheating)
• discard supernatent and leave tubes upside down on tissues
• replace caps and store two tubes upside down at -20^oC as backup
• miniprep remaining two using Qiagen miniprep kit (treat each as product of 2ml culture)
• gel run and extract to isolate RFI DNA from chromosomal/RNA contamination