Team:TU-Delft/Protocol 12

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Revision as of 13:50, 1 October 2013



Protocols

Our project deals with E.coli cells which sense Auto-inducing peptides (AIPs) from the Staphylococcus aureus and starts producing Antimicrobial peptides in order to kill the Staphylococcus aureus. Different protocols used during the project are described below.


Lysis Protocol

Procedure

  1. Grow the BL21(DE3) cells transformed with pT7 lysis cassette overnight in LB meduim with the right antibiotic, (in this case ampicillin or carbenicillin) .
  2. Make a 10X stock solution of IPTG by adding 10µL of the 1000X IPTG (1M) in 990 µL of LB with the right antibiotic.
  3. Make 1/50 times dilutions of the overnight grown culture. Wait untill the OD at 600nm reaches 0.4-0.6.
  4. Then make the below combinations into the 96 well plate for the plate reader to take readings.
1 2 3 4 5 6 7 8mM 9 10 11 12
No cells + 1mM 0.1mM 0.2m 0.3mM 0.4mM 0.5mM 0.6mM 0.7mM 0.8mM 0.9mM 1mM Cell + No IPTG
LB(µL) 90 94 93 92 91 90 89 88 87 86 85 95
Cells(µL) - 5 5 5 5 5 5 5 5 5 5 5
10X IPTG(µL) 10 1 2 3 4 5 6 7 8 9 10 -
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