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HighPure Plasmid Extraction Maxi Kit(SpinColumn)

Packing Specification：

Product Features:
1.The silica matrix membranes in the spin columns are all high-quality specially manufactured adsorption membranes, ensuring minimal variation in adsorption capacity between columns.
2.Eliminates the need for toxic reagents such as phenol and chloroform, as well as ethanol precipitation. Rapid and convenient: 0.2-0.5 mg of pure high-copy plasmid DNA can be quickly extracted from 100-200 mL of E. coli LB (Luria-Bertani) culture, with an extraction rate of approximately 80%.
3.The obtained plasmid has high yield, high supercoiled ratio, and good purity. It can be directly used for various molecular biology experiments such as restriction digestion, transformation, PCR, in vitro transcription, and sequencing.

Application Scope:
Suitable for the rapid extraction of 0.2-0.5 mg of pure high-copy plasmid DNA from 100-200 mL of E. coli LB culture, with an extraction rate of approximately 80%.

Operation Steps:
Tips:
Before first use, add the specified volume of absolute ethanol to the Wash Buffer WB bottle and Deproteinization Buffer PE bottle, mix thoroughly. Mark the corresponding box immediately after adding ethanol to avoid repeated additions!
Add all RNase A to Solution P1, mix well, and store at 2-8℃ after each use.
Precool Solution N3 on ice.

1.Take 150-200 mL of overnight bacterial culture, centrifuge at 12,000 x g (approximately 10,000 rpm) for 1-2 minutes, pour off the supernatant as much as possible, and collect the bacterial pellet. For collecting more than 50 mL of bacterial culture: Centrifuge to discard the supernatant, add more bacterial culture to the same 50 mL tube, and repeat Step 1 until sufficient bacterial pellet is collected.

2.Resuspend the bacterial pellet with 7.5 mL of Solution P1, pipette or vortex until completely suspended. Undissolved bacterial clumps will affect lysis, leading to low yield and purity.

3.Add 7.5 mL of Solution P2, gently invert up and down 8-10 times to fully lyse the bacteria, and incubate at room temperature for 3-5 minutes. Mix gently; do not shake vigorously to avoid shearing genomic DNA! The incubation time should not exceed 5 minutes to prevent plasmid damage. The bacterial solution should become clear and viscous. If it remains turbid, it may be due to excessive bacterial biomass and incomplete lysis; reduce the bacterial amount accordingly.

4.Add 7.5 mL of precooled Solution N3, immediately invert up and down gently 8-10 times. White flocculent precipitation will form when fully mixed. Incubate at room temperature or on ice for 5 minutes, centrifuge at 12,000 x g at 4℃ for 10 minutes, carefully transfer the supernatant, and avoid aspirating the floating white precipitate. Mix immediately after adding Solution N3 to avoid local precipitation of SDS. If there are still floating white precipitates in the supernatant, centrifuge again and transfer the supernatant.

5.Add 0.5 volumes of isopropanol (approximately 11 mL) to the supernatant obtained in the previous step, invert to mix thoroughly.

6.Load the mixture into the Spin DC Adsorption Column (placed in a collection tube) in multiple batches (≤10 mL per aliquot). Centrifuge at 12,000 x g for 1 minute, discard the waste liquid in the collection tube.

7.Add 10 mL of Deproteinization Buffer PE (check if absolute ethanol has been added!). Centrifuge at 12,000 x g for 1 minute and discard the waste liquid.

8.Add 10 mL of Wash Buffer WB (check if absolute ethanol has been added!). Centrifuge at 12,000 x g for 1 minute and discard the waste liquid. 

9.Repeat Step 8 once.

10.Place the Spin DC Adsorption Column back into the empty collection tube. Centrifuge at maximum speed (preferably >12,000 x g) for 2 minutes to dry residual ethanol on the membrane matrix. This step is critical to completely remove residual ethanol in the adsorption column, as ethanol residue inhibits downstream reactions and significantly reduces elution efficiency and plasmid yield.

Transfer the Spin DC Adsorption Column to a clean centrifuge tube. Add 1-2 mL of Elution Buffer EB (preheating the elution buffer in a 65-70℃ water bath can improve yield) to the center of the adsorption membrane. Incubate at room temperature for 2-5 minutes, then centrifuge at 12,000 x g for 2 minutes. For larger plasmid yield, reapply the eluate to the spin column and centrifuge for 2 minutes.Higher elution volume leads to higher elution efficiency. To obtain higher plasmid concentration, the elution volume can be appropriately reduced, but the minimum volume should not be less than 1 mL (excessively small volume reduces plasmid elution efficiency and yield).