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Team:Groningen/Notebook/10 August 2009
From 2009.igem.org
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| + | |||
| + | '''Ligation''' | ||
| + | |||
| + | (1:3) | ||
| + | :* 4 uL Ligase buffer | ||
| + | :* 2 ul T4 Ligase | ||
| + | :* 8 uL plasmid pSB1AC3-L digested with PstI and SpeI | ||
| + | :* 8 uL insert GVP restricted with XbaI and PstI | ||
| + | |||
| + | (1:6) | ||
| + | :* 4 uL Ligase buffer | ||
| + | :* 2 ul T4 Ligase | ||
| + | :* 8 uL plasmid pSB1AC3-M digested with PstI and SpeI | ||
| + | :* 4 uL insert GVP restricted with XbaI and PstI | ||
| + | |||
| + | ''Incubate:'' | ||
| + | :* 25°C 50min. | ||
| + | :* kept on ice for 10min. | ||
| + | |||
| + | '''Tranformation''' | ||
| + | :* add 10uL of the pSB1AC3-GVP ligation product to 50uL competent E.coli TOP10 cells. | ||
| + | ''Incubate:'' | ||
| + | :* 30 min @ ice | ||
| + | :* 50 sec 42°C | ||
| + | :* 2 min @ ice | ||
| + | :* add 800uL LB-medium | ||
| + | :* incubate for 1 h at 37°C | ||
| + | :* plate on LB-amp<sub>50</sub> plates | ||
===Transporters=== | ===Transporters=== | ||
Revision as of 13:16, 10 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
Restriction for Assembly
The vector [http://partsregistry.org/wiki/index.php?title=Part:pSB1AC3 pSB1AC3] containing the [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 high], [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23106 medium] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23109 low] constitutive promoters were cut with PstI and SpeI to create correct ends for insert of GVP biobrick [http://partsregistry.org/wiki/index.php?title=Part:BBa_I750016 BBa_I750016], which was cut with XbaI and PstI.
- 3 to 10μL plasmid in MQ (1.0μg)
- 6 to 13μL MQ (end volume of 20μL)
- 2μL Fast digest buffer
- 1μL PstI fast digest enzyme
- 1μL SpeI/XbaI fast digest enzyme
Restriction was kept at 37C for 30 min. and put on ice until used for gel purification.
Purification
Concentrations
| Plasmid | Conc. ng/μL | 260/280 | 260/230 | -20 box (michael | Restriction Control |
| pSB1AC3-H SpeI/PstI | ? | ? | ? | ? | ? |
| pSB1AC3-M SpeI/PstI | ? | ? | ? | ? | ? |
| pSB1AC3-L SpeI/PstI | ? | ? | ? | ? | ? |
| GVP XbaI/PstI restricted | ? | ? | ? | ? | Gel |
Ligation
(1:3)
- 4 uL Ligase buffer
- 2 ul T4 Ligase
- 8 uL plasmid pSB1AC3-L digested with PstI and SpeI
- 8 uL insert GVP restricted with XbaI and PstI
(1:6)
- 4 uL Ligase buffer
- 2 ul T4 Ligase
- 8 uL plasmid pSB1AC3-M digested with PstI and SpeI
- 4 uL insert GVP restricted with XbaI and PstI
Incubate:
- 25°C 50min.
- kept on ice for 10min.
Tranformation
- add 10uL of the pSB1AC3-GVP ligation product to 50uL competent E.coli TOP10 cells.
Incubate:
- 30 min @ ice
- 50 sec 42°C
- 2 min @ ice
- add 800uL LB-medium
- incubate for 1 h at 37°C
- plate on LB-amp50 plates
Transporters
Metal Accumulation
Vectors
Dry
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