Difference between revisions of "PhiX:DsDNA isolation"
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==Step One: phage amplification== | ==Step One: phage amplification== | ||
* prepare ~20ml of high titre phage (I have tried this from chemostat where pfu = 10<sup>9</sup>/ml) | * prepare ~20ml of high titre phage (I have tried this from chemostat where pfu = 10<sup>9</sup>/ml) | ||
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 37oC and follow until 0.3 < OD600nm < 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 37oC
- aliquot culture into four 50ml centrifuge tubes
- centrifuge @ 5000g for 20 minutes (4oC to avoid overheating)
- discard supernatent and leave tubes upside down on tissues
- replace caps and store two tubes upside down at -20oC 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
