Difference: ProtocolsGeneGorgingMarker (10 vs. 11)

Revision 112018-10-17 - JeffreyBarrick

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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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pJEB15 →Mal
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pJEB15 →Mal Amp<sup</
 
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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 g/ml Cam and 50 g/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 g/ml Cam and 100 g/ml Amp.
  • LB and TA plates with antibiotic. You will need LB + Cam + Amp, TA + Cam, TA + Amp, and TA plates.
 
    • 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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  1. Electroporate
  2. Pipette sample out of cuvette into original microfuge tube on ice. Add 500 l of room temperature SOC medium.
  3. Grow at 37C for 1 hr in a shaking incubator to induce antiobiotic expression.
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  1. Plate 100 l and 10 l + 90 l saline on two separate LB + Cam + Kan plates.
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  1. Plate 100 l and 10 l + 90 l saline on two separate LB + Cam + Amp plates.
 
  1. Grow plates overnight at 37C.

Day 2: Induce Gene-Gorging

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Day 6: Patch for Plasmid Loss

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  1. Patch 6-12 colonies from each plate on three LB (or TA/TM) plates with 20 g/ml Chloramphenicol, with Kanamycin, and - last - with no antibiotic.
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  1. Patch 6-12 colonies from each plate on three LB (or TA/TM) plates with Chloramphenicol, with Ampicillin, and - last - with no antibiotic.
 
  1. Grow plates overnight at 37C.

Day 7: Save a Freezer Stock of the New Strain

 
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