Team:Groningen/Notebook/15 July 2009
From 2009.igem.org
m (→GVP Cluster) |
m (→GVP Cluster) |
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:→ From left to right: 1kb Marker, | :→ From left to right: 1kb Marker, | ||
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+ | '''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=== | ===Transporters=== |
Revision as of 08:19, 15 July 2009
Wet
GVP Cluster
Restriction for Assembly
The vector (BBa_J61035) containing GVP cluster was cut with PstI and XbaI to create correct ends for insert in promotor vector (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 (BBa_J61002) containing promotor 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).
- → 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
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