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