User:Karmella Haynes/Notebook/ASU courses/2014/01/24

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  • Kozak plasmid minipreps

Kozak Plasmid Minipreps

  • For BME 100, spring 2014
  • Also for SynBERC Scholars/ Quanta "qOpenPCR" project

Day 1 - Tasks for Behzad:

  • Quick-transform Kozak miniprep into DH5α
  • Grow on 100μg/mL amp plates overnight. Include zero plasmid negative control

Day 2 - Tasks for Behzad

  • Pick 4 colonies, innoculate 4 cultures, 5 ml each
  • Grow overnight

Day 3 - Tasks for Behzad

  • Pellet the cells in the ancient table-top swing-bucket centrifuge
  • Very thoroughly (by vortexing and flicking and pipetting up and down) resuspend cells in appropriate amount of water
  • Proceed with miniprep
  • Measure DNA concentrations

Colony 1 2 3 4

Digest plasmids with restriction enzymes and gel electrophoresis (1% agarose) to confirm the assembled construct size

Plasmid DNA 3.0 μL
EcoRI1.0 μL
PstI1.0 μL
10x FastDigest buffer + green loading dye1.5 μL
dH2O8.5 μL
15.0 μL total
Incubate at 37°C for 10 minutes.

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