Plasmid Purification And Troubleshooting

Experiment Summary

Plasmids are small circular deoxyribonucleic acids (DNA) outside the chromosomes that replicate independently of chromosomal DNA. Although budding yeast and fission yeast can retain plasmids, the host of plasmids is almost always bacteria. This small circular DNA is widely used as a DNA vector in the fields of molecular biology, biochemistry, biotechnology, cell biology, etc. This means that plasmid purification/isolation is a very basic experiment in these research fields, and this experiment is performed almost every day in almost every laboratory. Plasmids are small circular DNA molecules that can replicate autonomously. They are mostly found in organisms such as bacteria and yeast. They are the most commonly used carriers in genetic engineering and are responsible for the important mission of transferring the target gene to the host cell.

Shaking Culture

1. Apply the cloned bacterial solution that has been identified and sequenced correctly on the LB solid plate (a culture medium for plasmid amplification).

2. Place in a 37℃ constant temperature incubator and culture for 12-17 hours until colonies grow.

3. Add 5ml of LB liquid culture medium containing antibiotics to a sterilized 15ml centrifuge tube and mark with numbers.

4. Pick a single clone bacterial group and place it in liquid culture medium, with 1 colony placed in each culture medium.

5. 37℃, 180rpm, shake culture overnight.

Harvest Bacteria And Lyse

1. Centrifuge the amplified bacterial solution, resuspend the bacterial solution according to the instructions, and transfer it to a 1.5 ml centrifuge tube.

2. Use a plasmid small-volume extraction kit to extract plasmids according to the instructions.

3. Centrifuge the bacteria at high speed for 1 minute and completely remove the supernatant.

4. Add 250 ul RB solution and fully suspend the bacteria on an oscillator.

5. Add 250 ul LB solution. Immediately turn upside down 10 times to lyse the bacteria, and place it at room temperature for 2 minutes.

6. Add 250 ul NB solution. Immediately turn upside down 10 times to fully neutralize it, and place it at room temperature for 2 minutes.

7. Centrifuge at room temperature, 1500 rpm, and high speed for 15 min.

8. Place the adsorption column in the collection tube, transfer the supernatant obtained by centrifugation to the adsorption column, and centrifuge at room temperature, 15000 rpm, and high speed for 30 s.

9. Discard the waste liquid, place the adsorption column in the collection tube, add 700 ul WB solution to the adsorption column, and centrifuge at 15,000 rpm for 30 s.

10. Repeat the previous operation.

11. Place the adsorption column in a clean 1.5 ml centrifuge tube, add 30-50 ul preheated Elution Buffer, place at room temperature for 2 min, and centrifuge at high speed for 1 min.

12. Electrophoresis of the sample: 1% agarose gel electrophoresis, with a sample volume of 2 ul. Observe under ultraviolet light, the brightest band is in the form of supercoil, which can indicate the purity of the extracted plasmid to a certain extent.

Plasmid Purification

1. Place the previously extracted plasmid in a 1.5 ml centrifuge tube and add 1/10 volume of 3 mol/L sterile sodium acetate solution.

2. Add 2 times the volume of anhydrous ethanol and place at -20℃ for precipitation for 4-6 h or overnight.

3. Centrifuge at 4℃, 14000 rpm, for 20 min.

4. Discard the supernatant and wash twice with 70% ethanol.

5. Air dry, or place on a clean bench for drying.

6. Add 200 ul of sterile aqueous solution and dry the resulting precipitate, which is the plasmid solution.

7. Measure the plasmid concentration and yield using a UV spectrophotometer.

Troubleshooting

1. Incomplete cell lysis

Cell density is too high: Reduce the culture volume.
Cell pellet is not completely resuspended: Properly resuspend the cell pellet with an appropriate buffer or solution before cell lysis.
Alkaline lysis step is insufficient: Check for salt precipitation in the lysis solution.

2. DNA or RNA contamination

Genomic DNA contamination: Do not vortex the cells too vigorously during the lysis and neutralization steps.
Lyophilized RNase is not completely dissolved: Dissolve the RNase as specified in the protocol.
RNA contamination: Add RNase to the resuspension solution before use.
Too many bacterial cells: Reduce the culture volume to avoid overloading the reaction or column.

3. Low or no yield

Culture volume is too high: Reduce the culture volume to increase the number of lysed cells.
Plasmid does not multiply: Use fresh bacterial cells for inoculation.
The effectiveness of kit components is reduced: Store kit components under appropriate conditions.
Washing solution is too concentrated: Cap the bottle tightly to avoid ethanol evaporation.

* For research use only. Not intended for any clinical use.

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