Team:Groningen/Notebook/15 July 2009

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Latest revision as of 12:45, 17 August 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) (329.7 ng/μL)
• 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
• 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) [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 BBa_J23100], and Empty slot

Gel Purification of GVP

"High Pure PCR Product Purification Kit" from [http://www.roche-applied-science.com 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)

[http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 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 [http://partsregistry.org/wiki/index.php/Part:BBa_I750016 GVP] into [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 BBa_J23100] [http://partsregistry.org/wiki/index.php?title=Part:BBa_J61002 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° with shaking 6 mm

- for mRFP1 excitation =590 nm

excitation =610 nm

Dry