User:Wilfred J. Poppinga/Notebook/Wilfreds Project/2009/08/19

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GVP

  • DONE Make picture of floating bacteria

MBP-ArsR

  • Make ON culture

Metal promotors

  • Look into follow up

Protocol #3

Restriction vectors

  • Mix
    • 2 μL Tango digest buffer (Fermentas)
    • 16 μL vector
    • 1 μL EcoRI (Non-fast digest)
    • 1 μL SpeI (Non-fast digest)
  • Incubate 1.5 h @ 37 °C
  • Put to 1% agarose (1xTBE)
    • Isolate ~3000 bp vector
    • Check for presence of cut out ccdB death gene (~600 bp)
  • Purify vector using gel purification kit
    • End volume 30 μL H2O (MilliQ)
    • Determine concentration using nanodrop
    • Store to 4 °C until ligation

Phosphorylation of 5' ends & hybridization [1]

  • (SENSE) Mix:


  • (ANTI-SENSE) Mix:
  • Incubate @ 37 °C for 1.5 hours.
  • Mix
    • 10 μL Sense mixture
    • 10 μL Anti-sense mixture
    • 3 μL 0.5 M NaCl
  • Place in boiling water for 3 min., and allow the reaction to cool to room temperature.

Ligation

  • Mix
    • 1 μL T4 ligase buffer
    • 7.5 μL restricted vector (purified from gel)
    • 1 μL annealing mix
    • 0.5 μL T4 ligase
  • Incubate
  • 1h RT
or
  • ON @ 4 °C

Transformation

  • Add 5 μL of ligation mixture to 50 μL of TOP10 chemically competent cells
  • Heatshock, 45 sec. 42 °C
    • + control: 1 μL pSB3K3-high or pSB1AC3-high plasmid, - control: 1 μL MilliQ
    • Alternatively a single cut plasmid can be taken as a ligation control
    • Alternative - control: 1 μL pSB1AC3 or pSB3K3 carrying ccdB deathgene
  • Incubate 1 h @ 37 °C, 200 RPM
  • Plate out on LB-agar + Kanamycin (30 μg/ml for pSB3K3) or Ampicillin (100 μg/mL for pSB1AC3)
    • Plate out 50 μL & 200 μL of cell suspension
  • Grow ON @ 37 °C

Checking transformations

  • See if - control is empty for functioning antibiotics and death gene
  • See how many colonies on + control for functioning competent cells
  • See how many selfclosers and compare to samples (>10x on sample vs. selfcloser)
  • If enough transformants, inoculate 3 - 5 colonies in an ON culture
    • Alternatively perform colony PCR

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FAKIN

  • TODO Mail Proost

FaBio

  • Finish paper

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