Team:Braunschweig/Protocols

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     <h2><a href="#Miniprep">Minipreparation of Plasmid DNA</a></h2>
     <h2><a href="#Miniprep">Minipreparation of Plasmid DNA</a></h2>
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Miniprep was done with Plasmid Miniprep Kit I by Peqlab (High Copy) following the manufacturer’s protocol.<br>
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First 2 mL of the cell suspension were spun down for 10 minutes (5000g).<br>
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For the alkaline lysis of the DNA the Pellet was resuspended in 250 μL Solution I (RNAseA). 250 μL of Solution II and 350 μL of solution III were consecutively added. As final step of the alkaline lysis the mixtures was centrifuged at 10.000 g for 10 minutes.<br>
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Afterwards the PerfectBindColumn was loaded with 750 μL lysate and then centrifuged for 1 minute (10000g). Subsequently the DNA was washed with 500 μL PW plasmid buffer and centrifuged again for 1 minute (10.000g). The DNA was the precipitated with the Ethanol containing wash buffer and separated by another centrifugation (1min, 10000g) and dried.<br>
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At last the DNA was eluted with 50 μL elution buffer, which was pre-heated to 60°C, and incubated for 5 min. The eluted DNA was separated from the matrix by a final centrifugation step (1 min, 5000g).
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Revision as of 23:35, 2 October 2013

Protocols

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In this section you will find detailed protocols of experimental procedures.

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