Team:Groningen/Notebook/10 July 2009

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TestTTTTTTTT
'''Glycerol Stocks'''
'''Glycerol Stocks'''

Revision as of 09:57, 10 July 2009

Igemhomelogo.png

TestTTTTTTTT

Glycerol Stocks

From the o.n. cultures of E.coli TOP10 with plasmids BBa_J23109, BBa_J23100 and BBa_J23106...

Plasmid Purification

Plasmid isolation was performed on the cultures of promotor containing plasmids in 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.
  • 350μL of Neutralisation Solution was added and the tubes inverted.
  • Cell debri was pelleted by centrifugation at full speed for 1 min.

Concentration of Plasmids

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

BBa_J23109 eluted in MQ

  • ng/μL
  • (260/280)
  • (260/230)

BBa_J23100 eluted in MQ

  • ng/μL
  • (260/280)
  • (260/230)

BBa_J23106 eluted in MQ

  • ng/μL
  • (260/280)
  • (260/230)

Restriction analysis

The plasmids containing GVP were cut with EcoRI and XbaI fast digest enzymes. The double digestion should result in two fragments of 1400 and 8000 bp in size.

  • μL MQ
  • μL plasmid in MQ
  • 2μL Fast digest buffer
  • 1μL EcoRI fast digest enzyme
  • 1μL XbaI fast digest enzyme

The cups were incubated in a heat block at 37°C for 15 min. followed by adding 4μL 6x loading buffer for gel electroforese.

Gel electroforese

10μL of each sample was loaded on a 2% agarose gel with EtBr and a 1kb ladder was used (see picture).


April
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May
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June
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29 30
July
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August
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31
September
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October
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November
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