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Team:Groningen/Notebook/10 July 2009
From 2009.igem.org
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{{Team:Groningen/Notebook/Day/Header}} | {{Team:Groningen/Notebook/Day/Header}} | ||
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'''Glycerol Stocks''' | '''Glycerol Stocks''' | ||
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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. | ||
| - | ''BBa_J23109 eluted in MQ'' | + | ''[http://partsregistry.org/wiki/index.php?title=Part:BBa_J23109 BBa_J23109] eluted in MQ'' |
* ng/μL | * ng/μL | ||
* (260/280) | * (260/280) | ||
* (260/230) | * (260/230) | ||
| - | ''BBa_J23100 eluted in MQ'' | + | ''[http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 BBa_J23100] eluted in MQ'' |
* ng/μL | * ng/μL | ||
* (260/280) | * (260/280) | ||
* (260/230) | * (260/230) | ||
| - | ''BBa_J23106 eluted in MQ'' | + | ''[http://partsregistry.org/wiki/index.php?title=Part:BBa_J23106 BBa_J23106] eluted in MQ'' |
* ng/μL | * ng/μL | ||
* (260/280) | * (260/280) | ||
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'''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. | + | 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 | * 2μL Fast digest buffer | ||
* 1μL EcoRI fast digest enzyme | * 1μL EcoRI fast digest enzyme | ||
Revision as of 12:39, 10 July 2009
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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.
- 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).
PCR HmtA Mut1/Mut2
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