AlanFung:Protocol/Plasmid DNA amplification: Difference between revisions
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==Grow cells on LB-Plates== | ==Grow cells on LB-Plates== | ||
*Thaw transformation competent E.Coli cells on ice | *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 DNA solution (<5ul per 50ul cells) to 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 agar plates containing appropriate antibiotic and incubate overnight at 37C | |||
*Perform transformation with 1-2ul of diluted shRNA plasmid | *Perform transformation with 1-2ul of diluted shRNA plasmid | ||
*Plate out the transformants on LB-Chloramphenicol plates | *Plate out the transformants on LB-Chloramphenicol plates |
Revision as of 23:23, 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
- 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 DNA solution (<5ul per 50ul cells) to 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 agar plates containing appropriate antibiotic and incubate overnight at 37C
- 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