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