No matter whether you're extracting small or large amounts of plasmid DNA, this is indispensable.

　　 Centrifuge It plays a very important role in plasmid DNA extraction and has a complex operational procedure.

　　Because plasmid DNA serves as a vector for genetic recombination and gene transfer, it is crucial in genetic engineering research.

　　Therefore, plasmid DNA is frequently used in routine cell experiments. Nowadays, kits make the process extremely convenient. After culturing the bacterial cells, you can concentrate and extract the plasmid from multiple tubes at once, yielding a substantial amount of plasmid DNA. This DNA can be stored at -20℃ for future use in experiments such as restriction enzyme digestion and transformation. You can even run a gel electrophoresis in advance—so long as the DNA doesn't degrade, it remains usable, eliminating the need to repeatedly culture and extract fresh samples each time you plan to use it.

　　There are two common methods for extracting plasmid DNA:

　　I. Plasmid Mini Prep

　　Add the antibiotic (ampicillin at a concentration of 1:1000) to the previously prepared LB liquid medium. Take a 15 ml centrifuge tube and add 5 ml of the liquid medium containing the antibiotic.

　　Using the “Xiao Bai Tips,” pick out single colonies from the plate, and pipette the small tips into a 15 ml centrifuge tube. Incubate at 30℃ with shaking at 180 rpm for more than 5 hours, until the liquid medium becomes turbid.

　　Take a clean 1.5 ml EP tube, and pipette 1.5 ml of bacterial culture from the 15 ml centrifuge tube (the volume can be adjusted up or down depending on the turbidity of the bacterial suspension). Centrifuge at 12,000 rpm for 3 minutes.

　　Discard the supernatant and add 250 μl of TIANGEN Plasmid Mini Prep Kit (Cat. No. DP103-03, Lot No. 6123) P1 solution. Shake vigorously to ensure thorough mixing.

　　Add 250 μl of P2 solution, and gently invert the tube 6–8 times (to avoid DNA shearing) to ensure thorough lysis of the bacterial cells. (The total duration of this step should not exceed 5 minutes to prevent over-lysis and potential damage to the plasmid.)

　　Add 350 μl of P3 solution to the EP tube, and immediately gently invert the tube 6–8 times to thoroughly mix the contents. At this point, you should observe a white flocculent precipitate. Centrifuge at 12,000 rpm for 3 minutes. (After adding P3, mix immediately to prevent localized precipitation. If there is still a tiny white precipitate in the supernatant, you can centrifuge again and then carefully collect the supernatant.)

　　Place the adsorption column CP3 into the collection tube. (Before the experiment, treat the adsorption column with the equilibration buffer BL to maximize activation of the silica-based membrane and improve the yield. It is best to use the BL-treated adsorption column on the same day; prolonged storage may affect its performance.) Transfer the supernatant into the adsorption column CP3, being careful not to draw up any precipitate. Centrifuge at 12,000 rpm for 30–60 seconds. Discard the waste liquid from the collection tube and place the adsorption column back into the collection tube.

　　Optional step: Add 500 µl of protein-depleting solution PD to CP3, centrifuge at 12,000 rpm for 30–60 seconds, discard the waste liquid from the collection tube, and place the adsorption column into the collection tube. (This step is recommended if the host strain is an end A+ strain such as TG1, BL21, HB101, JM series, or ET12567, as these strains contain high levels of nucleases; however, this step can be omitted if the host strain is an end A- strain such as DH5α or TOP10.)

　　Add 600 μl of wash buffer PW (containing anhydrous ethanol) to the CP3 column. Centrifuge at 12,000 rpm for 30–60 seconds. Discard the waste liquid from the collection tube and place the adsorption column into the collection tube.

　　Repeat step 9.

　　Centrifuge at 12,000 rpm for 2 minutes to remove any residual wash buffer remaining in the adsorption column.

　　Place the CP3 adsorption column into a clean 1.5 ml EP tube. Add 50–100 μl of elution buffer EB to the middle of the adsorption membrane, let it sit at room temperature for 2 minutes, then centrifuge at 12,000 rpm for 2 minutes. Collect the plasmid solution into the EP tube. (To increase the yield of plasmid, you can re-add the solution to the CP3 adsorption column, let it sit at room temperature for 2 minutes, centrifuge at 12,000 rpm for 2 minutes, and collect the plasmid solution into the EP tube again. Alternatively, you can preheat the EB buffer to 65–70℃ in advance.)

　　Measure the concentration and run the gel to assess the quality of the plasmid extract.

　　2. For plasmid large-scale extraction, it is generally advisable to perform a small-scale plasmid extraction beforehand to confirm the quality of the plasmid.

　　Take a clean conical flask and add 100 ml of the LB liquid medium prepared in Step 1. Then, pipette 10 μl of the bacterial culture prepared in Step 2 into the conical flask. Incubate at 30℃ with shaking at 180 rpm overnight, until the liquid medium becomes turbid.

　　Preserving strains: To preserve strains using 15% glycerol, add 350 μl of bacterial suspension and 150 μl of 50% glycerol to a 1.5 ml EP tube, mix thoroughly, and store at -80℃.

　　Transfer the remaining bacterial suspension from the conical flask into a 50 ml centrifuge tube, and centrifuge at 4°C and 8,000 rpm for 5 minutes to collect the bacterial pellet. You can use the same tube multiple times.

　　Follow the steps outlined in the instructions for the selected DNA extraction kit; the specific steps may vary depending on the kit used.

　　After extraction, measure the concentration and run the gel (since plasmid molecules generally have a large molecular weight, use a low-concentration agarose gel with large-sized markers).

　　The above is a summary of the applications of centrifuges in plasmid DNA extraction. If you’d like to learn more about product applications, please stay tuned to Shanghai Luxiangyi Centrifuge Instrument Co., Ltd.