Haynes Lab:Notebook/Engineering PC-TFs/2012/02/01

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QIAprep Spin Minprep Kit

  • Pellet 1 - 5 mL bacterial overnight culture by centrifugation at >8000 rpm for 3 min at room temperature
  • Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to microcentrifuge tube
  • Add 250 µl Buffer P2 and mix by inverting tube 6 times until solution is clear.
  • Add 350 µl Buffer N3 and mix by inverting the tube 6 times.
  • Centrifuge for 10 minutes at 13,000 rpm
  • Apply the supernatant liquid to the QIAprep spin by decanting. Centrifuge for 30-60 second and discard the flow-through.
  • Wash the QIAprep column by adding .75 ml Buffer PE
  • Centrifuge for 60 seconds and discard the flow through.
  • Centrifuge for 1 min to remove residual wash buffer.
  • Place the QIAprep column in a clean 1.5 ml tube. To remove the DNA, add water to the center of the QIA spin columns, and centrifuge for 2 min.

Transformation of Linker Genes

  • Transformations: Linker genes from Genscript (4 total)

> Synthesized DNA from Genscript (red screw-cap tubes)

  1. PL rigid
  2. PL rigid 4
  3. PL flex
  4. PL flex 4

Note: these parts arrived in the pUC57 vector: [1]

> Warm five 100 μg/mL Amp agar plates at 37 °C
> Thaw fresh tube of DH5α Turbo cells on ice
> Resuspend invisible DNA pellet (stock DNA) in 10 μL dH2O
> Add 0.5 μL stock DNA to 10 μL dH2O in 0.5 mL tubes
> Add 30 μL of DH5α Turbo cells to DNA + dH2O
--> Include #5 tube, water only, no DNA (negative control)
> Incubate cells + DNA on ice for 5 min.
> Label pre-warmed plates
> Transfer cells + DNA onto agar
> Add 10 - 15 sterile glass beads, shake, discard beads
> Incubate plates at 37 °C overnight