Team:Groningen/Notebook/13 August 2009
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Revision as of 08:42, 13 August 2009 by Verhoeven1981 (Talk | contribs)
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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