Team:Groningen/Notebook/21 August 2009
From 2009.igem.org
Wet
GVP Cluster
Ligation
(1:?)
- 2 uL Ligase buffer
- 1 ul T4 Ligase
- 2 uL plasmid BBa_J61002 digested with EcoRI and SpeI
- 1 uL insert oligo's with pMetal+RBS EcoRI/SpeI ends
Incubate:
- 25°C 60min.
- 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-amp100 plates
Over Night Cultures
Transporters
PCR on pBAD plasmid with primers F2 rev has worked. cut out and isolated 11 ug/ul Used in 3 PCRs, (pcr1)F1 mut1rc, (pcr2)Rev mut2rc and F1 Rev.
Metal Accumulation
Vectors
Dry
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