Team:Groningen/Notebook/21 August 2009

From 2009.igem.org

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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

April
MTWTFSS
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27 28 29 30
May
MTWTFSS
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4 5 6 7 8 9 10
11 12 13 14 15 16 17
18 19 20 21 22 23 24
25 26 27 28 29 30 31
June
MTWTFSS
1 2 3 4 5 6 7
8 9 10 11 12 13 14
15 16 17 18 19 20 21
22 23 24 25 26 27 28
29 30
July
MTWTFSS
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6 7 8 9 10 11 12
13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30 31
August
MTWTFSS
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3 4 5 6 7 8 9
10 11 12 13 14 15 16
17 18 19 20 21 22 23
24 25 26 27 28 29 30
31
September
MTWTFSS
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7 8 9 10 11 12 13
14 15 16 17 18 19 20
21 22 23 24 25 26 27
28 29 30
October
MTWTFSS
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5 6 7 8 9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27 28 29 30 31
November
MTWTFSS
            1
2 3 4 5 6 7 8
9 10 11 12 13 14 15
16 17 18 19 20 21 22
23 24 25 26 27 28 29
30