Team:Groningen/Notebook/10 July 2009

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Revision as of 12:48, 10 July 2009

Glycerol Stocks

From the o.n. cultures of E.coli TOP10 with plasmids [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23109 BBa_J23109], [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 BBa_J23100] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23106 BBa_J23106] glycerol stocks were made by adding 250μL of 87% sterile glycerol to 750μL of culture. The cells were frozen in liquid nitrogen and stored at -80°C.

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.

[http://partsregistry.org/wiki/index.php?title=Part:BBa_J23109 BBa_J23109] eluted in MQ

• 61.4 ng/μL
• 1.89 (260/280)
• 2.23 (260/230)

[http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 BBa_J23100] eluted in MQ

• 96.1 ng/μL
• 1.88 (260/280)
• 2.22 (260/230)

[http://partsregistry.org/wiki/index.php?title=Part:BBa_J23106 BBa_J23106] eluted in MQ

• 55.4 ng/μL
• 1.84 (260/280)
• 2.22 (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 plasmids containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23109 BBa_J23109], [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 BBa_J23100], and [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23106 BBa_J23106] were cut with EcoRI and PstI fast digest enzymes. The double digestion should result in two fragments of 1400 and 8000 bp in size.

• 0μL (8μL) MQ
• 16μL (8μL) plasmid in MQ
• 2μL Fast digest buffer
• 1μL EcoRI fast digest enzyme
• 1μL PstI 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 (25μL for GVP) was loaded on a 2% agarose gel with EtBr and a 1kb ladder was used (see picture).

Gel Purification of GVP

A standard kit for PCR-product purification was used for gel purification...

PCR HmtA Mut1/Mut2