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Team:Groningen/Notebook/14 July 2009
From 2009.igem.org
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===GVP Cluster=== | ===GVP Cluster=== | ||
| - | [[Image:Plasmid Miniprep Kit Flow Protocol.gif|thumb| | + | [[Image:Plasmid Miniprep Kit Flow Protocol.gif|thumb|150px| www.sigmaaldrich.com]] |
'''Plasmid Purification''' | '''Plasmid Purification''' | ||
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* 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. | * 350μL of Neutralisation Solution was added and the tubes inverted. | ||
| - | * Cell debri was pelleted by centrifugation at full speed for 1 min. | + | * Cell debri was pelleted by centrifugation at full speed for 10 min. |
| - | * | + | * Column was prepared by adding 500μL Column Preparation Solution and centrifuging for 1 min. full speed |
| + | * The lysate was transfered to the column and centrifuged for 1 min. full speed | ||
| + | * Column was washed with both washing solutions | ||
| + | * Plasmids were eluted with 30μL MQ and stored in the fridge | ||
'''Concentration of Plasmids''' | '''Concentration of Plasmids''' | ||
Revision as of 09:12, 14 July 2009
[http://2009.igem.org/Team:Groningen http://2009.igem.org/wiki/images/f/f1/Igemhomelogo.png]
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Wet
GVP Cluster
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 5mL 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 10 min.
- Column was prepared by adding 500μL Column Preparation Solution and centrifuging for 1 min. full speed
- The lysate was transfered to the column and centrifuged for 1 min. full speed
- Column was washed with both washing solutions
- Plasmids were eluted with 30μL MQ and stored in the fridge
Concentration of Plasmids
The concentration of isolated plasmid was determined with the use of a nano-drop.
GVP eluted in MQ
- ng/μL
- (260/280)
- (260/230)
Transporters
Dry
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