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Team:Groningen/Notebook/13 August 2009
From 2009.igem.org
(Difference between revisions)
(New page: {{Team:Groningen/Notebook/Day/Header}} ==Wet== ===GVP Cluster=== :→ {{todo}} make fresh LB-agar medium :→ {{todo}} inocculate o.n. cultures for glycerol stocks :→ {{todo}} ligatio...) |
(→GVP Cluster) |
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| Line 7: | Line 7: | ||
:→ {{todo}} make fresh LB-agar medium | :→ {{todo}} make fresh LB-agar medium | ||
:→ {{todo}} inocculate o.n. cultures for glycerol stocks | :→ {{todo}} inocculate o.n. cultures for glycerol stocks | ||
| - | :→ {{ | + | :→ {{done}} ligation of GVP into vectors |
:→ {{todo}} transformation of E.coli TOP10 with ligation product | :→ {{todo}} transformation of E.coli TOP10 with ligation product | ||
| + | |||
| + | '''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-amp<sub>50</sub> plates | ||
===Transporters=== | ===Transporters=== | ||
Revision as of 08:42, 13 August 2009
[http://2009.igem.org/Team:Groningen http://2009.igem.org/wiki/images/f/f1/Igemhomelogo.png]
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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
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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