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Transfection

Plasmid Isolation. Transfection. Using this as a reference for the DNA plasmid isolation protocol will help you along the way. Transfect plasmid into cells Grow up E. Coli cells. Plasmid Isolation. Centrifuge cells in high-speed centrifuge for 20 minutes at 4000 rpm.

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Transfection

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  1. Plasmid Isolation Transfection Using this as a reference for the DNA plasmid isolation protocol will help you along the way • Transfect plasmid into cells • Grow up E. Coli cells

  2. Plasmid Isolation • Centrifuge cells in high-speed centrifuge for 20 minutes at 4000 rpm

  3. 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.

  4. 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)

  5. 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

  6. 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

  7. 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

  8. Place sample with Plasmid DNA and Cesium Chloride in dialysis tubing • Dialyze overnight in TE at 4°C

  9. 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

  10. Check DNA • Use the Nanodrop ND-1000 to check on the concentration and purity of your sample

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