Team:Lyon-INSA-ENS/Realisation/Protocols/DNA-purification

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A final DNA concentration of up to 0.4µg/µL can be expected, if eluting a high-copy plasmid with 500µL of Buffer TE. eluates from low-copy vectors or dilute samples can be concentrated by spinning in a centrifuge under vacuum, or ethanol precipitation. Low-copy plasmids that have been amplified in the presence of chloramphenicol should be treated as high-copy plamids when choosing the appropriate culture volume.
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A final DNA concentration of up to 0.4µg/µL can be expected, if eluting a high-copy plasmid with 500µL of Buffer TE.  
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<h6> Procedure </h6>
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1. Pick a single colony from a freshly streaked selective plate and inoculate a starter culture of 2-5mL LB meduim containing the appropriate selective antibiotic. Incubate for approx. 8h at 37°C with vigorious shaking (approx. 300 rpm).
1. Pick a single colony from a freshly streaked selective plate and inoculate a starter culture of 2-5mL LB meduim containing the appropriate selective antibiotic. Incubate for approx. 8h at 37°C with vigorious shaking (approx. 300 rpm).
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2. Dilute the starter 1/500 to 1/1000 into selective LB medium. For high-copy plasmids inoculate 50mL.   
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Revision as of 09:32, 18 July 2011





Plasmid or Cosmid DNA Purification Using HiSpeed Plasmid Midi Kits




This protocol is for preparation of up to 200µg of high- or low-copy plasmid or cosmid DNA using the HiSpeed Plasmid Midi Kit.

A final DNA concentration of up to 0.4µg/µL can be expected, if eluting a high-copy plasmid with 500µL of Buffer TE.





Procedure


1. Pick a single colony from a freshly streaked selective plate and inoculate a starter culture of 2-5mL LB meduim containing the appropriate selective antibiotic. Incubate for approx. 8h at 37°C with vigorious shaking (approx. 300 rpm).

2. Dilute the starter 1/500 to 1/1000 into selective LB medium. For high-copy plasmids inoculate 50mL.



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