Madhadron:ElectroporateSmegmatis

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===Materials===
 
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* Sterile water with 0.05% tween-80
 
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* 7H9 medium
 
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* LB plates with: no marker; 50μg/mL hygromycin and 25μg/mL kanamycin; 25μg/mL kanamycin
 
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===Directions===
 
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# Grow 100mL culture of ''M. smegmatis'' overnight to A<sub>600</sub> = 0.6-0.8.
 
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# Centrifuge culture at 3,000rpm for 10 minutes at 4&deg;C.  Aspirate supernatant and resuspend in same volume of sterile water + 0.05% tween-80.  Repeat.
 
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# Centrifuge once more, and resuspend in 2mL of just sterile water (no tween).
 
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# Electroporate 400&mu;L plus DNA with 2.5kV, 1k&Omega; 25&mu;F, and immediately transfer electroporated slurry to 5mL recovery medium.
 
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# Incubate recovery medium shaking at 37&deg;C for at least 3.5 hours.
 
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# Centrifuge recovery medium at 3,000 rpm for 10 minutes.  Resuspend in 300&mu;L PBS + 0.05% tween-80 and plate out, 100&mu;L per plate, on blank (no antibiotics), kanamycin, and hyg+kan.
 
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===Notes===
 
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* Positive controls: an integrating plasmid (pMV361); no DNA with electroporation on plain LB; no DNA without electroporation on plain LB.
 
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* Negative controls: no DNA with and without electroporation on antibiotic plates.
 

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