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

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(Labwork)
(Labwork)
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==Labwork==
==Labwork==
'''Plating of ''E.coli TOP10'' cells'''
'''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.
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".
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.
* 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.
* 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.
* 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'''
'''Concentration of Plasmids'''
 +
The concentration of isolated plasmid was determined with the use of a nano-drop.
The concentration of isolated plasmid was determined with the use of a nano-drop.
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* 1.91 (260/280)
* 1.91 (260/280)
* 2.78 (260/230)
* 2.78 (260/230)
 +
 +
''Terminator eluted in MQ''
 +
* 35.3 ng/μL
 +
* 1.74 (260/280)
 +
* 1.97 (260/230)
 +
 +
''Terminator eluted in elution buffer''
 +
* 38.8 ng/μL
 +
* 1.89 (260/280)
 +
* 1.93 (260/230)
'''Restriction analysis'''
'''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.
 +
 +
* 6μL  MQ
 +
* 10μL 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'''
'''Gel electroforese'''
 +
 +
10μL of each sample was loaded on a 2% agarose gel with EtBr and a 1kb ladder was used (see picture).
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Revision as of 06:25, 9 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.
  • 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.

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)

Terminator eluted in MQ

  • 35.3 ng/μL
  • 1.74 (260/280)
  • 1.97 (260/230)

Terminator eluted in elution buffer

  • 38.8 ng/μL
  • 1.89 (260/280)
  • 1.93 (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.

  • 6μL MQ
  • 10μL 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).



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