Team:Groningen/Notebook/26 August 2009

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Wet

GVP Cluster

Restriction for Assembly

The vector pSB1AC3 containing the LAC and pBAD inducible promoters were cut with PstI and SpeI to create correct ends for insert of GVP biobrick BBa_I750016, which was cut with XbaI and PstI.

  • 3 to 16μL plasmid in MQ (1.0μg)
  • 13μL MQ (end volume of 20μL)
  • 2μL Fast digest buffer
  • 1μL PstI fast digest enzyme
  • 1μL SpeI/XbaI fast digest enzyme

Restriction was kept at 37C for 30 min. and put on ice until used for gel purification.

Purification

Concentrations

Plasmid Conc. ng/μL 260/280 260/230 -20 box (michael Restriction Control
pSB1AC3-LAC SpeI/PstI ? ? ? ? ?
pSB1AC3-pBAD SpeI/PstI ? ? ? ? ?
GVP XbaI/PstI restricted ? ? ? ? Gel

Ligation

(1:6)

  • 4 uL Ligase buffer
  • 2 ul T4 Ligase
  • 8 uL plasmid pSB1AC3-LAC digested with PstI and SpeI
  • 8 uL insert GVP restricted with XbaI and PstI

(1:6)

  • 4 uL Ligase buffer
  • 2 ul T4 Ligase
  • 8 uL plasmid pSB1AC3-pBAD digested with PstI and SpeI
  • 4 uL insert GVP restricted with XbaI and PstI

Incubate:

  • 25°C 50min.
  • 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-amp50/chlo20 plates

Transporters

Metal Accumulation

MBP-ArsR

  • Transform ligation with inducible promotors
  • Prepare glycerol stock without promotors

Vectors

Dry

April
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June
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July
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August
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September
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October
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November
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