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

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)

Transformations
> 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