Team:Groningen/Notebook/26 August 2009
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Wet
GVP Cluster
Restriction for Assembly
The vector pSB1AC3 containing the LAC and pBAD inducible promoters were cut with PstI and SpeI to create correct ends for insert of GVP biobrick BBa_I750016, which was cut with XbaI and PstI.
- 3 to 16μL plasmid in MQ (1.0μg)
- 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
A "Agarose Gel DNA Extraction Kit" Standard Protocol from Roche Applied Science.
- → In step ten, 30μL MQ was added to the dry pellet and incubated at room temperature.
Concentrations
Plasmid | Conc. ng/μL | 260/280 | 260/230 | -20 box (michael | Restriction Control |
pSB1AC3-LAC SpeI/PstI | ? | ? | ? | ? | ? |
pSB1AC3-pBAD SpeI/PstI | ? | ? | ? | ? | ? |
GVP XbaI/PstI restricted | ? | ? | ? | ? | Gel |
Ligation
(1:6)
- 4 uL Ligase buffer
- 2 ul T4 Ligase
- 8 uL plasmid pSB1AC3-LAC 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-pBAD 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
- 90 sec 42°C
- 2 min @ ice
- add 800uL LB-medium
- incubate for 1 h at 37°C
- plate on LB-amp50/chlo20 plates
Transporters
Metal Accumulation
MBP-ArsR
- Transform ligation with inducible promotors
- Prepare glycerol stock without promotors
Vectors
Dry
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