AlanFung:Protocol/Plasmid DNA amplification: Difference between revisions
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*Aliquot 50-100ul of cells into chilled 17x100mm polypropylene tubes | *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 | *Unused cell may be frozen (For optimal recovery, refreeze cells in a dry ice/ ethanol bath prior to -70C storage | ||
*Add | *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 | *Incubate on ice for 30mins | ||
*Place tubes in 42C water bath for 30 to 45 seconds without shaking | *Place tubes in 42C water bath for 30 to 45 seconds without shaking | ||
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*Dilute transformation reactions to 1ml by addition of 900-950ul SOC medium | *Dilute transformation reactions to 1ml by addition of 900-950ul SOC medium | ||
*Shake the tube 200rpm for 60 minutes at 37C | *Shake the tube 200rpm for 60 minutes at 37C | ||
*Plate by spreading 5-200ul of cell transformation mixture on LB | *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== | ==Grow cells in LB-Broth== |
Latest revision as of 00:09, 21 September 2010
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