Plasmid Isolation Using this as a reference for the DNA plasmid isolation protocol will help you...
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Transcript of Plasmid Isolation Using this as a reference for the DNA plasmid isolation protocol will help you...
Plasmid IsolationUsing this as a reference for the DNA plasmid isolation protocol will help you
along the way
Transfection
• Transfect plasmid into cells
• Grow up E. Coli cells
Plasmid Isolation
• Centrifuge cells in high-speed centrifuge for 20 minutes at 4000 rpm
• Lyse cells and centrifuge in high-speed centrifuge for 10 minutes at 3500 rpm
Supernatant contains the DNA. The white solid is the cell lysate; membrane, organelles, proteins.
• Transfer supernatant to a 50 ml tube
• Add isopropyl alcohol and spin for 10 minutes at 3500 rpm in high speed centrifuge
Pellet is DNA from the sample (E.coli and plasmid mixed)
• Drain supernatant and resuspend pellet in TE
• Add 7.73g Cesium Chloride and transfer to 9ml ultracentrifuge tube
• Add 350 μl Ethidium Bromide and fill tube with TE
• Centrifuge in Ultracentrifuge overnight at 55,000 rpm at 23°C
• Remove cap and insert needle and syringe right below band and remove band
This band is the plasmid DNA
• Place band in a 50ml tube and add an equal volume of TE
• Add a volume of Isoamyl alcohol, shake, and allow to separate
• Remove top pink layer with alcohol and Ethidium Bromide and repeat till clear
• Place sample with Plasmid DNA and Cesium Chloride in dialysis tubing
• Dialyze overnight in TE at 4°C
• After dialysis place in 50ml tube and add 1/10 volume NaCl and 2x volume Ethanol
• Centrifuge at 4°C for 15 minutes at 4000 rpm
• Remove supernatant and rinse with 95% Ethanol
• Resuspend in dH20
Check DNA
• Use the Nanodrop ND-1000 to check on the concentration and purity of your sample