Lab 8: Plasmid Isolation and Restriction Mapping
Activity A1:
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To each of two 1.5 mL microcentrifuge tubes, add 1.5 mL of culture
Spin tube for 5 min at 6000 rpm
Decant supernatant and dispose in biowaste baskets
Resuspend the two pellets in 125 ul of buffer P1 (mix completely)
Pool both 125ul suspension into 1.5 ml micro tubes
Add 250 ul of buffer P2 and mix by inverting
Add 350 ul of buffer N3 and mix quickly
Spin tube for 10 minutes at 13,000 rpm in microcentrifuge, the use a pipette to remove as
much supernatant as possible. Place in an ion exchange column
a. Spin the column for 60 sec at 13,000 rpm
9. Add 500 ul of buffer pb and spin for 60 sec at 13,000 rpm
10. Add 750 ul of buffer PE and spin for 60 sec at 13,000
11. Place the column into the empty collection tube and spin for 60 sec at 13,000 rpm
12. Add 50 ul of buffer EB to the column and spin for 60 sec at 13,000 rpm. Then discard the
spin column and keep the microcentrifuge tube
13. Combine the contents of your tube with just one other group in the class
Activity B1
1. Recover pGLo plasmid DNA
2. Label 8 microcentrifuge tubes 1-8
3. Place sterile water, 10X buffer, and tubes of EcoR1, EcoRV, and Scal restriction
enzymes in an ice bucket
4. The table below shows the ingredients to add to each of the 8 tubes. Once added, incubate
for 60 min in the 37 degree dry bath
Activity C1:
1. Set up casting tray
2. Obtain molten 1% agarose in 1xTBE with EtBr from 55C water bath and pour 100 mL
into casting degree
3. After gel has set place into electrophoresis chamber
4. Pour enough 1X TBE buffer to fill both chambers just enough to cover gel and well depth
of one mm
Activity C2:
Lab 8: Plasmid Isolation and Restriction Mapping
1. Add 4 ul of loading dye to pGlo DNA digests and mix. Spin the samples in
microcentrifuge
2. Load your samples in wells of the gel from left to right in the following order:
1,2,3,4,5,6,7,8, ladder. Load 6 ul of ladder in the first and last lanes, load 24 ul of samples
1-8
3. Run the gel at 100 Volts for 60 minutes
4. Turn off power supply, place gel under UV, take digital picture, use picture in your lab
report