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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.