IGEM:metu/2009/Notebook/wound dressing/2009/10/01

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01.10.2009

1. 10:00 --- We separeted LB+Amp+ ChL+ Bacteria with ABC and also LB+ Amp+ Bacteria without ABC into microcentrifuge tubes that contain 2 ml. 10 microcentrifuge tubes were prepared for each of them.

2. 10:30 --- Storage of pEGF-TLiA-pEcPrt-DEF (with ABC transporter) and pEGF-TLiA (without ABC ).

3. We measured OD of tubes. We wanted to catch value of OD is between 2-2,5.

11:40 -- first measurement of OD

pEGF-TLiA-pEcPrt-DEF (with ABC) : 0.8
pEGF-TLiA (without ABC )        : 0.8

16:45 -- second measurement of OD

pEGF-TLiA-pEcPrt-DEF (with ABC) : 2.081
pEGF-TLiA (without ABC )        : 2.044

4. 18:00 -- We made holes (diameter of holes are 3 mm) in the plates. In one plate, there were two holes. One hole was for pEGF-TLiA-pEcPrt-DEF (with ABC transporter) and the other hole was for pEGF-TLiA (without ABC ). We add 10 microliter of supernatant into these holes(or zones).

Also we add just 1 microliter of pellet on plate without making zone.

We put these plates into incubation ,25 °C ,48 hours.

5. The rest of tubes were centrifugated at 4000 rpm and 5 minutes.

  • After centrifuge, we took the supernatant and add protein inhibitor over supernatant .
  • We put the tubes ,that we will use tomorrow , in -20 °C. Other tubes were put in -80 °C.