Team:Groningen/Notebook/21 August 2009

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.

PCR worked. Isolated and pcr to get the final construct.

Metal Accumulation

 * PCR to amplify fMT, SmtA with pre-RBS
 * Redo PCR from yesterday as the PCR didnt proceed (hot start was manually)
 * Run on gel and excise bands.


 * Ligate pSB1AC3 with MymT, fMT and SmtA
 * Do restriction of the vector and the inserts with EcoRI and SpeI.
 * Do PCR clean-up kit to purify from the restriction enzymes.
 * Check concentration and ligate o/n @ 4&deg;C 100ng in a 3x overexcess of insert.