Team:Groningen/Notebook/15 July 2009

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Revision as of 08:19, 15 July 2009

Wet

GVP Cluster

Restriction for Assembly

The vector ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J61035 BBa_J61035]) containing [http://partsregistry.org/wiki/index.php/Part:BBa_I750016 GVP] cluster was cut with PstI and XbaI to create correct ends for insert in promotor vector ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J61002 BBa_J61002]).

• 10μL MQ
• 6μL plasmid in MQ (2μg)
• 2μL Fast digest buffer
• 1μL PstI fast digest enzyme
• 1μL XbaI fast digest enzyme

The vector ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J61002 BBa_J61002]) containing promotor [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 BBa_J23100] was cut with SpeI and PstI.

• 4μL MQ
• 10μL plasmid in MQ
• 2μL Fast digest buffer
• 1μL SpeI fast digest enzyme
• 1μL PstI fast digest enzyme

The cups were incubated in a heat block at 37°C for 30 min. followed by adding 4μL 6x loading buffer for gel electroforesis.

Gel electroforesis

24μL of each sample was loaded on a 1% agarose gel with EtBr and a 1kb ladder was used (see picture).

[[Image:]]

→ From left to right: 1kb Marker,

Gel Purification of GVP

"High Pure PCR Product Purification Kit" from [www.roche-applied-science.com Roche] was used for gel purification.

• 200mg of gel containing the desired fragment was dissolved in 600μL binding buffer by heating to 60°C for 10 min. and carefully vortexing.
• 300μL isopropanol was added to the tube and vortexed
• dissolved gel was transfered to the column and centrifuged for 1 min.
• column was washed with 500μL Wash Solution, centrifuged for 1 min., 200μL Wash solution was added and centrifuged for 1 min.
• 15μL MQ was applied to the column, incubated for 3 min. at room temperature and centrifuged for 1 min. at full speed.

Concentration of GVP-vector

The concentration of isolated cut vector of GVP with EcoRI and XbaI was determined with the use of a nano-drop.

GVP-vector eluted in MQ

•  ? ng/μL
•  ? (260/280)
•  ? (260/230)

→

Transporters

Dry