Team:Groningen/Notebook/13 August 2009
From 2009.igem.org
(Difference between revisions)
(→GVP Cluster) |
(→GVP Cluster) |
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Line 9: | Line 9: | ||
:→ {{done}} ligation of GVP into vectors | :→ {{done}} ligation of GVP into vectors | ||
:→ {{todo}} transformation of E.coli TOP10 with ligation product | :→ {{todo}} transformation of E.coli TOP10 with ligation product | ||
+ | |||
+ | '''Bouyancy Test''' | ||
+ | |||
+ | To test the effect of the metals Cu, Zn and Ar on bouyancy cells containing the metal sensitive promoters + GVP (msGVP) construct are grown in liguid media containing a range of different metal concentrations. As controls there is a reaction tube without Cu, Zn or Ar and reaction tubes with cells that contain the pSB1AC3 vector instead of msGVP. | ||
+ | |||
+ | Procedure: | ||
+ | * Grow cells o.n. in liquid culture (LB, Amp 100 ug/ml) containing the correct amount of inducer (see table metal concentrations). | ||
+ | * Centrifuge: 1000rpm, ~20 min | ||
+ | * Remove medium and resuspend in saline (0.15 mM NaCl solution) | ||
+ | * The tubes are then kept at room temp. without shaking | ||
+ | |||
+ | |||
+ | '''Metal Concentrations''' | ||
+ | {|cellpadding="2" cellspacing="1" border="1" | ||
+ | |Cu | ||
+ | |0 | ||
+ | |0.01 mM | ||
+ | |0.1 mM | ||
+ | |0.5 mM | ||
+ | |1 mM | ||
+ | |2 mM | ||
+ | |- | ||
+ | |Zn | ||
+ | |0 | ||
+ | |2 uM | ||
+ | |4 uM | ||
+ | |8 uM | ||
+ | |10 uM | ||
+ | |15 uM | ||
+ | |- | ||
+ | |Ar | ||
+ | |0 | ||
+ | |0.01 mM | ||
+ | |0.1 mM | ||
+ | |? | ||
+ | |? | ||
+ | |? | ||
+ | |} | ||
+ | |||
'''Ligation''' | '''Ligation''' |
Revision as of 12:37, 13 August 2009
Wet
GVP Cluster
- → TODO make fresh LB-agar medium
- → TODO inocculate o.n. cultures for glycerol stocks
- → DONE ligation of GVP into vectors
- → TODO transformation of E.coli TOP10 with ligation product
Bouyancy Test
To test the effect of the metals Cu, Zn and Ar on bouyancy cells containing the metal sensitive promoters + GVP (msGVP) construct are grown in liguid media containing a range of different metal concentrations. As controls there is a reaction tube without Cu, Zn or Ar and reaction tubes with cells that contain the pSB1AC3 vector instead of msGVP.
Procedure:
- Grow cells o.n. in liquid culture (LB, Amp 100 ug/ml) containing the correct amount of inducer (see table metal concentrations).
- Centrifuge: 1000rpm, ~20 min
- Remove medium and resuspend in saline (0.15 mM NaCl solution)
- The tubes are then kept at room temp. without shaking
Metal Concentrations
Cu | 0 | 0.01 mM | 0.1 mM | 0.5 mM | 1 mM | 2 mM |
Zn | 0 | 2 uM | 4 uM | 8 uM | 10 uM | 15 uM |
Ar | 0 | 0.01 mM | 0.1 mM | ? | ? | ? |
Ligation
(1:3)
- 4 uL Ligase buffer
- 1 ul T4 Ligase
- 9 uL plasmid pSB1AC3-H digested with PstI and SpeI
- 9 uL insert GVP restricted with XbaI and PstI
(1:3)(2x)
- 4 uL Ligase buffer
- 1 ul T4 Ligase
- 12 uL plasmid pSB3K3-H/L digested with PstI and SpeI
- 5 uL insert GVP restricted with XbaI and PstI
Incubate:
- 25°C 60min.
- kept on ice for 10min.
Tranformation
- add 10uL of the pSB1AC3-H-GVP and pSB3K3-H/L-GVP ligation products to 50uL competent E.coli TOP10 cells.
Incubate:
- 30 min @ ice
- 90 sec 42°C
- 2 min @ ice
- add 800uL LB-medium
- incubate for 1 h at 37°C
- concentrate in 100uL LB-medium
- plate on LB-amp50 plates
Transporters
Metal Accumulation
Vectors
Dry
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