Transfection

1
Transfection
Plasmid Isolation
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 23C
• 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 4C

9

• After dialysis place in 50ml tube and add 1/10
  volume NaCl and 2x volume Ethanol
• Centrifuge at 4C 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