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Team:Groningen/Notebook/26 August 2009
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Wet
GVP Cluster
- → TODO Restriction of GVP and pSB1AC3-Lac/pBAD for assembly
- → TODO Gel purification of wanted fragments and nanodrop
- → TODO Clone GVP cluster behind Lac and Arabinose promoter for expression tests in pSB1AC3 plasmid
- → TODO Test promoter strenght compared to BBa_J23101 promoter (Sven)
- → TODO Enter sequences of constructs to Sandbox
Restriction for Assembly
The vector [http://partsregistry.org/wiki/index.php?title=Part:pSB1AC3 pSB1AC3] containing the LAC and pBAD inducible 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 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
- → From left to right: 1kB ladder, pSB1AC3-LAC ,Empty Slot, pSB1AC3-pBAD-AraC , Empty Slot, GVP
- → The band for the pSB1AC3-LAC vector was expected to be at ~3700bp, but instead it was at ~2500bp.
- → Comparison with the gel of Frans (constructed pSB1AC3-LAC) explained the band at a lower position (also seen on his gel), but not why it is so small. One option is use of pSB3K3 plasmid instead of pSB1AC3 plasmid, this would still give a band of ~3300bp and not ~2500bp!
A "Agarose Gel DNA Extraction Kit" [http://www.roche-applied-science.com/pack-insert/1696505a.pdf 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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