21 Lab Protocol: Plasmid Isolation and Bacterial Transformation
Plasmid DNA Isolation from Bacteria
- Work in a group of two. Each group should obtain one Eppendorf tube containing a bacterial pellet. Place in tube rack.
- Add 100 µl of P1 Buffer to the tube and resuspend pellet completely using a vortex or by pipetting. The purpose of P1 buffer is to resuspend the cells. Plasmid DNA remains inside the cells.
- Add 100 µl of P2 Buffer and mix by inverting and/or rotating the tube 2 – 4 times. Cells are completely lysed when the solution appears clear, purple, and viscous. Proceed to the next step within 1-2 minutes. The purpose of P2 buffer is to lyse the cells and to allow the plasmid DNA to escape.
- Add 200 µl of P3 Buffer and mix gently but thoroughly. Do not vortex. The sample will turn yellow when neutralization is complete. P3 buffer removes contaminating cellular debris as a pellet in step 6 and neutralizes the pH so plasmid DNA will attach to column in step 7.
- Allow the lysate to incubate at room temperature for 1-2 minutes.
- Centrifuge sample(s) for 2 minutes.
- Place a Zymo-Spin™ IIN column in a Collection Tube and transfer the supernatant from Step 6 into the Zymo-Spin™ IIN column. When pipetting the supernatant be careful not to disturb the green pellet to avoid transferring any cellular debris to the column.
- Centrifuge the Zymo-Spin™ IIN/Collection Tube assembly for 30 seconds. The plasmid DNA sticks to the column and contaminants flow through the column into the collection tube.
- Discard the flow-through in the Collection Tube, making sure the flow-through does not touch the bottom of the column. Return the Zymo-Spin™ IIN column to the Collection Tube.
- Add 100 µl of Endo-Wash Buffer to the column and centrifuge for 30 seconds. Discard flowthrough. This wash step and the next wash step remove impurities from the plasmid DNA which remains attached to column.
- Add 200 µl of Plasmid Wash Buffer to the column. Centrifuge for 1 minute.
- Transfer the column into a clean 1.5 ml microcentrifuge tube and then add 30 µl of DNA Elution Buffer to the column. Centrifuge for 30 seconds to release the plasmid DNA into the microcentrifuge tube.
Analysis of Plasmid DNA Yield by Agarose Gel Electrophoresis
Note: agarose gels contain the mutagen Ethidium bromide; therefore, only the instructor should handle gels wearing gloves.
- Pipet 10 µL loading buffer/dye to 12 locations on parafilm.
- Each group will mix 6 µL plasmid with the appropriate drop of loading buffer on parafilm. A diagram should be labeled indicating where each group placed their plasmid DNA.
- Each group will load 12 µL of their sample into the appropriate well of a 1% agarose gel submerged in 1X TBE running buffer.
- Load size ladder into an empty well.
- Once all wells have been loaded, connect electrodes (be sure positive electrode is on far side of gel from wells).
- Turn on power and allow migration to occur for 30 minutes. Turn off gel.
- Instructor (wearing gloves) will transfer gel to gel documentation system to visualize the results.
- Photograph the results. We will only want to use tubes known to contain plasmid DNA in the bacterial transformation procedure.
Preparation and Transformation of Competent E. coli Cells
- Scrape cells from petri dish using sterile pipet tip or toothpick. Mix cells in 250 mL ice cold, 50 mM CaCl2 and place in ice bucket. Incubate for 10 minutes.
- Scrape cells from petri dish using sterile pipet tip or toothpick. Mix cells in 250 mL ice cold, 50 mM CaCl2 and place in ice bucket. Incubate for 10 minutes.
- Transfer tubes to 42o C water bath for 50 seconds (heat shock). Return tubes to ice bucket. Incubate 5 minutes.
- Add 300 µL of LB growth medium, mix gently, and incubate 15 minutes at 37o C to allow bacterial cells to recover and begin expressing the antibiotic resistance gene.
- Spread 100 mL cells onto each of three different LB agar plates: – ampicillin, + ampicillin, + ampicillin + arabinose. Two groups will share each plate.
- Incubate plates overnight at 37o C to allow cells to grow, divide and form colonies.
Note that all bacterial cells are expected to grow on the petri dish without ampicillin, and only transformed cells will grow on plates with ampicillin. Arabinose is a sugar that will allow expression of the green fluorescent protein in transformed cells.
