Team:Groningen/Notebook/15 July 2009

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

Mix:
 * 1 μL T4 Ligase buffer
 * 1 μL Vector (J23100 restriction fragment, 14.2 ng/μL)
 * 7.6 μL Insert (GVP gene cluster, 33.5 ng/μL)
 * 0.5 μL T4 Ligase

Method:
 * The mixture is incubated 30 min, 26°C
 * To inactivate Ligase 10 min 70°C

Transformation of E.coli TOP10 cells with GVP + J23100


 * 2 μL Ligation mix added to cells from -80°C glycerol stock
 * 30 min incubation on ice
 * 5 min heat shock 37°C
 * 5 min on ice
 * Cells added to 3 ml Ty in a reaction tube
 * Incubation 1 h in waterbath shaker 37°C


 * The cells are plated on Ty agar (100 μg/ml)
 * On one plate 200 μL is streaked out. The rest is concentrated (centrifugation: 1 min, 14600rpm) in 200 μL and also plated.
 * Incubation o/n 37°C

Transporters
We decided to make MasterMixes of our own. All but 1uL template, 2uL primers and 1uL taq for 25uL reactions.

Vectors

 * overnight cultures were diluted 100, 200 or 400 x
 * 200 ul of culture per well
 * Measured for 16 h
 * - every 15 min
 * - @ 37&deg; with shaking 6 mm
 * - for mRFP1 excitation =590 nm
 * excitation =610 nm