AlanFung:Protocol/Plasmid DNA amplification

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Plasmid DNA Amplification[edit]

For pRFP-C-RS based HuSH vector from Origene[edit]

Dilution of Plasmid solution[edit]

  • Add 50ul of dH2O into each tube
  • Vortex the tubes to resuspend the DNA
  • Pipette 1ul of this soultion to another tube
  • Add 99ul H2O Concentration of the DNA solution should be 1ng/ul
  • Notes: Store plasmid solution at -20C

Grow cells on LB-Plates[edit]

  • Thaw transformation competent E.Coli cells on ice
  • Mix cells gently by lightly flicking the tube
  • Aliquot 50-100ul of cells into chilled 17x100mm polypropylene tubes
  • Unused cell may be frozen (For optimal recovery, refreeze cells in a dry ice/ ethanol bath prior to -70C storage
  • Add 2ul of diluted shRNA plasmid solution to 50ul cell suspension and gently swirl tube for a few second to mmix
  • Incubate on ice for 30mins
  • Place tubes in 42C water bath for 30 to 45 seconds without shaking
  • Place tube again on ice for 2 mins
  • Dilute transformation reactions to 1ml by addition of 900-950ul SOC medium
  • Shake the tube 200rpm for 60 minutes at 37C
  • Plate by spreading 5-200ul of cell transformation mixture on LB-Chloramphenicol plates containing appropriate antibiotic and incubate overnight at 37C until colonies appear

Grow cells in LB-Broth[edit]

  • The following day inoculate single bacterial colonies into 5ml of LB-KChloramphenicol and grow them overnight

Purify DNA plasmids[edit]

Purify DNA plasmids form the culture using a miniprep DNA isolation kit / Qiagen Endo-free maxiprep kit(Vector Core recommended)

Prepare plasmids for packaging[edit]

  • Resuspend the DNA in 50ul of TE solution
  • Determine the concentration of the samples and make sure it meets the core requirements