Streptomyces:Protocols/Conjugation: Difference between revisions
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==Protocols - | ==Protocols - Conjugation== | ||
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[[Category:Protocol]] | |||
[[Category:Streptomyces]] | |||
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| || Preparing the recipient cells || ~15 minutes | | || Preparing the recipient cells || ~15 minutes | ||
|- | |- | ||
| || Plating the conjugant mix || ~20 minutes | | || Plating the conjugant mix || ~20 minutes | ||
|- | |- | ||
| || Whole Conjugation || ~1 hour 30 minutes | | || Whole Conjugation || ~1 hour 30 minutes | ||
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| || colspan="2" | | |||
---- | |||
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| || Overlay preparation || ~5 minutes | | || Overlay preparation || ~5 minutes | ||
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'''Uses''' | '''Uses''' | ||
<br/> | <br/> | ||
Transfer a cloned plasmid/cosmid into Streptomyces to | Transfer a construct (cloned plasmid/cosmid) into Streptomyces to knock-out, knock-in, complement or over-express a gene. | ||
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# Plate parallels if necessary – see notes. | # Plate parallels if necessary – see notes. | ||
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Overlay: | *Overlay: | ||
**After an overnight incubation, the conjugation plates need to be overlaid with antibiotics, to select for successful exconjugants. Typically, the antibiotics used are Nalidixic Acid and whatever antibiotic the conjugated construct confers resistance to; normally Apramycin. | |||
** After an overnight incubation, the conjugation plates need to be overlaid with antibiotics, to select for successful exconjugants. Typically, the antibiotics used are Nalidixic Acid and whatever antibiotic the conjugated construct confers resistance to; normally Apramycin. | |||
** Create a 500µL solution per plate using sdH<sub>2</sub>O and the usual concentrations of antibiotics. | ** Create a 500µL solution per plate using sdH<sub>2</sub>O and the usual concentrations of antibiotics. | ||
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Latest revision as of 05:59, 14 August 2007
Protocols - Conjugation
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Intergeneric Conjugation and OverlayDescription
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