Team:Groningen/Notebook/15 July 2009
From 2009.igem.org
m (→GVP Cluster) |
m (→GVP Cluster) |
||
Line 9: | Line 9: | ||
* 10μL MQ | * 10μL MQ | ||
- | * 6μL plasmid in MQ (2μg) | + | * 6μL plasmid in MQ (2μg) (329.7 ng/μL) |
* 2μL Fast digest buffer | * 2μL Fast digest buffer | ||
* 1μL PstI fast digest enzyme | * 1μL PstI fast digest enzyme | ||
Line 17: | Line 17: | ||
* 4μL MQ | * 4μL MQ | ||
- | * | + | * 12μL plasmid in MQ (155.4 ng/μL) |
* 2μL Fast digest buffer | * 2μL Fast digest buffer | ||
* 1μL SpeI fast digest enzyme | * 1μL SpeI fast digest enzyme |
Revision as of 10:01, 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) (329.7 ng/μL)
- 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
- 12μL plasmid in MQ (155.4 ng/μL)
- 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 (devided over two slots) with EtBr and a 1kb ladder was used (see picture).
- → From left to right: Empty slot, 1kb Marker, Empty slot, HmtA PCR reaction, Empty slot, (2x) GVP, Empty slot, (2x) BBa_J23100, and Empty slot
Gel Purification of GVP
"High Pure PCR Product Purification Kit" from Roche was used for gel purification.
- ~200mg of gel containing the desired fragment was dissolved in 500μL binding buffer by heating to 60°C for 10 min. and carefully vortexing.
- 250μL 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-cluster eluted in MQ
- 33.5 ng/μL
- 1.65 (260/280)
- 0.77 (260/230)
BBa_J23100 + vector eluted in MQ
- 14.2 ng/μL
- 1.78 (260/280)
- 1.01 (260/230)
- → Concentrations are just high enough for a ligation reaction to be performed, some additional tuning of the procedure is required!!
Ligation of GVP into BBa_J23100 vector
For ligation an amount of 10ng cut vector is required, and a 6:1 molar ratio insert to vector
Transporters
Dry
|
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
|
|
|