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| META TOPICPARENT |
name="ProtocolList" |
Gene Gorging Marker Mutations |
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| Plasmid |
Marker |
| pJEB11 |
→Ara– |
| pJEB12 |
→Ara+ |
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- Antibiotic stock solutions of Chloramphenicol (Cam) and Kanamycin (Kan).
- Final concentrations of antibiotics in all media (liquid or plates) are 25 µg/ml Cam and 50 µg/ml Kan.
- LB and TA plates with antibiotic. You will need LB + Cam + Kan, TA + Cam, TA + Kan, and TA plates.
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- Antibiotic stock solutions of Chloramphenicol (Cam) and Ampicillin (Amp).
- Final concentrations of antibiotics in all media (liquid or plates) are 25 µg/ml Cam and 100 µg/ml Amp.
- LB and TA plates with antibiotic. You will need LB + Cam + Amp, TA + Cam, TA + Amp, and TA plates.
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- Note: The protocol refers to TA plates throughout. If you are not using the Ara marker, substitute the proper sugar, e.g. make TM plates if using the maltose marker.
- 20% L-Arabinose stock. Dissolve 10 g in 50 ml of ddH20 and filter sterilize or autoclave.
- Electrocompetent cells of the parent strain.
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- Electroporate
- Pipette sample out of cuvette into original microfuge tube on ice. Add 500 µl of room temperature SOC medium.
- Grow at 37°C for 1 hr in a shaking incubator to induce antiobiotic expression.
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- Plate 100 µl and 10 µl + 90 µl saline on two separate LB + Cam + Kan plates.
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- Plate 100 µl and 10 µl + 90 µl saline on two separate LB + Cam + Amp plates.
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- Grow plates overnight at 37°C.
Day 2: Induce Gene-Gorging |
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Day 6: Patch for Plasmid Loss |
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- Patch 6-12 colonies from each plate on three LB (or TA/TM) plates with 20 µg/ml Chloramphenicol, with Kanamycin, and - last - with no antibiotic.
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- Patch 6-12 colonies from each plate on three LB (or TA/TM) plates with Chloramphenicol, with Ampicillin, and - last - with no antibiotic.
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- Grow plates overnight at 37°C.
Day 7: Save a Freezer Stock of the New Strain |
