AlanFung:Protocol/Plasmid DNA amplification: Difference between revisions

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


==Prepare plasmids for packaging==
==Prepare plasmids for packaging==
*Resuspend the DNA in 50ul of TE solution  
*Resuspend the DNA in 50ul of TE solution  
*Determine the concentration of the samples and make sure it meets the core requirements
*Determine the concentration of the samples and make sure it meets the core requirements

Revision as of 22:00, 20 September 2010

Plasmid DNA Amplification

For pRFP-C-RS based HuSH vector from Origene

Dilution of Plasmid solution

  • 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

  • Thaw transformation competent E.Coli cells on ice
  • Perform transformation with 1-2ul of diluted shRNA plasmid
  • Plate out the transformants on LB-Chloramphenicol plates
  • Incubate overnight at 37C, until colonies appear

Grow cells in LB-Broth

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

Purify DNA plasmids

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

Prepare plasmids for packaging

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