User:Michael Verhoeven/Notebook/iGEM 2009 M. Verhoeven Parts/2009/07/07: Difference between revisions

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==Labwork==
==Labwork==
* plates with colonies
'''Plating of ''E.coli TOP10'' cells'''
The plates with transformed ''E.coli TOP10'' cells contained between 5-50 colonies for the non-diluted plates, depending on which plasmid was transformed into the cells. To increase the number and size of the colonies, the plates were stored at 37°C for an additional 24 hours.


* plasmid purification
'''Plasmid Purification'''
Plasmid isolation was performed on the cultures of GVP and Terminator containing cells with the "Sygma-Aldrich™ GenElute™ Plasmid Miniprep Kit".
* From each tube 2mL of culture was collected in a 2mL cup, and the cells were pelleted by centrifugation for 1 min. at max. speed and the supernatant discarded.
* Cells were resuspended in 200μL Resuspension Solution by up and down pipetting.
* To the mixture 200μL Lysis Solution was added, mixed by inverting the cup, and stored at room temperature for 5 minutes.
*  


* concentration
'''Concentration of Plasmids'''
The concentration of isolated plasmid was determined with the use of a nano-drop.


* Restriction analysis
''GVP eluted in MQ''
* 88.2 ng/μL
* 1.83 (260/280)
* 2.08 (260/230)


* Gel electroforese
''GVP eluted in elution buffer''
* 92.2 ng/μL
* 1.91 (260/280)
* 2.78 (260/230)
 
'''Restriction analysis'''
 
'''Gel electroforese'''


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Revision as of 00:50, 8 July 2009

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Labwork

Plating of E.coli TOP10 cells The plates with transformed E.coli TOP10 cells contained between 5-50 colonies for the non-diluted plates, depending on which plasmid was transformed into the cells. To increase the number and size of the colonies, the plates were stored at 37°C for an additional 24 hours.

Plasmid Purification Plasmid isolation was performed on the cultures of GVP and Terminator containing cells with the "Sygma-Aldrich™ GenElute™ Plasmid Miniprep Kit".

  • From each tube 2mL of culture was collected in a 2mL cup, and the cells were pelleted by centrifugation for 1 min. at max. speed and the supernatant discarded.
  • Cells were resuspended in 200μL Resuspension Solution by up and down pipetting.
  • To the mixture 200μL Lysis Solution was added, mixed by inverting the cup, and stored at room temperature for 5 minutes.

Concentration of Plasmids The concentration of isolated plasmid was determined with the use of a nano-drop.

GVP eluted in MQ

  • 88.2 ng/μL
  • 1.83 (260/280)
  • 2.08 (260/230)

GVP eluted in elution buffer

  • 92.2 ng/μL
  • 1.91 (260/280)
  • 2.78 (260/230)

Restriction analysis

Gel electroforese



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