Plasmid DNA Amplification and Isolation | Day 2: Growing a Liquid Culture скачать в хорошем качестве

Plasmid DNA Amplification and Isolation | Day 2: Growing a Liquid Culture

Protocol: Bacterial Culturing from Stabs and Plasmid DNA Isolation Protocol Prepared by: Michael Sandway, Haoyu Xu This three-day protocol is intended for use with bacterial stabs already containing the plasmid, rather than purified plasmid DNA. Day One: 1. Autoclave 100 mL LB broth in an autoclave-safe glass bottle and let cool 2. Sterilize the work area with a solution of 70% ethanol. 3. Take the bacterial stab (containing the plasmid) and one agar plate (coated with the corresponding antibiotics specified in the bacterial stab) out of 4°C storage and leave it on the workbench. 4. Label the plastic cover of the plates with the name of the plasmid being cultured, the bacteria strain, your initials, and date. For example: Tube #1 – LifeAct-GFP – NiCo21(DE3) – AB – 11/11/2025. 5. Using a sterile inoculation loop, gently scrape a small amount of bacteria from the surface of the stab medium, and perform quadrant streaking on the agar plate. 6. After streaking, place the plates back in an inverted position inside the incubator to incubate about 24 h at 37°C. Day Two: 7. Add the right type of antibiotics to the broth and mix. Use recommended concentration from the place where the bacterial stab was ordered. 8. Using an inoculation loop, take a single colony off the plate and put it in broth. 9. Put broth in 37°C shaking incubator for 16-20 h (18 h is preferable) at 180 rpm. Day Three: 10. Aliquot inoculated broth into sixteen 15 mL tubes containing 5 mL each (amount of tubes and broth depend on DNA extraction protocol). Plasmid purification using Spin Miniprep Kit. This kit uses a method called alkaline lysis to separate the small, circular plasmid DNA from the large, tangled genomic DNA and other cell debris. Notes before starting: • Optional: Add LyseBlue reagent to Buffer P1 at a ratio of 1 to 1000. • Add the provided RNase A solution to Buffer P1, mix and store at 2–8°C. • Add ethanol (96–100%) to Buffer PE before use (see bottle label for volume). • Unless otherwise specified, all centrifugation steps are carried out at 13,000 rpm (~17,900 x g) in a conventional table-top microcentrifuge. 11. Pellet Cells: Spin down the sixteen 5 mL of bacterial culture in a centrifuge at 4200 rpm for 5 min. 12. Resuspend (Buffer P1): Remove the liquid from the 15 mL tubes. Resuspend pelleted bacterial cells from every two 15 mL tubes in 250 µL Buffer P1 and transfer to a microcentrifuge tube. Sixteen 15 mL tubes of pelleted bacterial cells will be resuspended into eight microcentrifuge tubes. 13. Lysis (Buffer P2): In each microcentrifuge tube, add 250 µL Buffer P2 about 1/8 inch above the P1 solution. Mix thoroughly by gently inverting the microcentrifuge tube 4–6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 min. If using LyseBlue reagent, the solution will turn blue. 14. Neutralization (Buffer N3): Add 350 µL Buffer N3 to each of the microcentrifuge tube, and mix immediately, gently, and thoroughly by inverting the microcentrifuge tubes 4–6 times. Do not shake the tubes. If using LyseBlue reagent, the solution will turn colorless. 15. Centrifugation: Centrifuge at 13,000 rpm (~17,900 x g) for 10 min. 16. Bind to Column: For each microcentrifuge tube, remove the supernatant (about 900 µL) and transfer it to the spin column by pipetting. Centrifuge for 1 min and discard the flow-through. 17. Wash (Buffers PB): Recommended: Wash the spin column by adding 500 µL Buffer PB. Centrifuge for 1 min and discard the flow-through. 18. Wash (Buffers PE): Wash the spin column by adding 750 µL Buffer PE. Centrifuge for 1 min and discard the flow-through. Transfer the spin column to the collection tube. 19. Centrifuge for 1 min to remove residual wash buffer. 20. Elute (Buffer EB): Place the column in a clean 1.5 mL microcentrifuge tube. To elute DNA, add 50 µL Buffer EB (10 mM TrisCl, pH 8.5) or water to the center of the spin column, let stand for 1 min, and centrifuge for 1 min. 21. Check the density of purified plasmid DNA and label the tubes with the plasmid name and concentration. 22. Temporarily store the purified plasmid DNA at -20°C before moving to -80°C for long-term storage.