Difference: ProtocolsPhageLysisTiming (2 vs. 3)

Revision 32018-06-07 - ColinBrown

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META TOPICPARENT name="ProtocolList"

Measuring Lysis Timing of T7 Phage

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Changed:
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  • 250mL flasks
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  • 2-3x 250mL flasks
  • Shaking water bath
 
  • For plating phages:
    • LB Plates with w/ appropriate supplements and antibiotics
    • LB Top Agar (LB + 0.8% Agar)
    • Test tubes
    • 55C Water bath or heat block
Changed:
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    • 37C Incubator
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    • 30C or 37C Incubator
 

Procedure:

  1. Add 500uL of an overnight culture of the E. coli host to 9.5mL of LB in a 250mL flask
Changed:
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  1. Allow to grow for 1h @ 37C, 200RPM until culture density is 1-2e8 cells / mL (OD ~ 0.2-0.3)
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  1. Allow to grow for 1.5h @ 30C, 200RPM until culture density is 1-2e8 cells / mL (OD ~ 0.2-0.3)
 
  1. Add 5e7 phages and incubate 5min without shaking at 37C* (may need to be shorter for some strains?) *30C for nsAA evolved strains
Changed:
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  1. Dilute 100uL of culture into 900uL of media in an eppendorf tube, then transfer 10uL into 10mL fresh media pre-warmed to 37C* (10000x dilution total) (may need to adjust volumes?)
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  1. Dilute 100uL of culture into 900uL of media in an eppendorf tube, then transfer 10uL into 10mL fresh media pre-warmed to 37C* (10000x dilution total)
 
  1. Plate samples at 1min timepoints from 5-15min (may depend on strain)
    • plate 10uL of each dilution as described in Determining Phage Titers
    • Probably don't need to set up dilutions for these (?)
 
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