Team:Groningen/Notebook/21 August 2009

From 2009.igem.org

Latest revision as of 16:44, 21 August 2009

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.

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°C 100ng in a 3x overexcess of insert.

Vectors

Dry