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HighPure Plasmid Extraction Midi Kit(SpinColumn)

Packing Specification：

Product Introduction:

This kit uses an improved SDS-alkaline lysis method to lyse cells. The silica matrix membrane in the spin column selectively binds plasmid DNA in the solution under high-salt and low-pH conditions. Impurities and other bacterial components are removed through deproteinization solution and wash buffer. Finally, pure plasmid DNA is eluted from the silica matrix membrane using low-salt and high-pH elution buffer.

Product Features:
1.The silica matrix membranes in the spin columns are all specially manufactured adsorption membranes imported from world-renowned companies, ensuring minimal variation in adsorption capacity between columns and good reproducibility. It overcomes the drawback of unstable membrane quality in domestic kits.
2.Unique deproteinization buffer formulation efficiently removes residual nucleases. It can easily eliminate nucleases even from strains with high nuclease content, such as JM series and HB101, effectively preventing plasmid degradation by nucleases.
3.Rapid and convenient: Eliminates the need for toxic reagents such as phenol and chloroform, as well as ethanol precipitation. The obtained plasmid has high yield and purity, and can be directly used for various molecular biology experiments such as restriction digestion, transformation, PCR, in vitro transcription, and sequencing.

Application Scope:
Suitable for medium-scale plasmid preparations (mini preparations) from 5-15 mL of bacterial culture.

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 P3 on ice.
1.Take 5-15 mL of overnight bacterial culture, centrifuge at 9,000 rpm for 1-2 minutes, pour off the supernatant as much as possible, and collect the bacterial pellet.

2.Resuspend the bacterial pellet with 500 µL of Solution P1, vortex until completely suspended, and transfer all to a 2 mL centrifuge tube. 
Undissolved bacterial clumps will affect lysis, leading to low yield and purity.

3.Add 500 µL of Solution P2, gently invert up and down 4-7 times to fully lyse the bacteria, and incubate at room temperature for 4 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 at this time. If there are few bacteria, proceed to the next step once it becomes clear and viscous; exact 5-minute incubation is not required.

4.Add 700 µL of precooled Solution P3, immediately invert up and down gently 4-7 times. White flocculent precipitation will form when fully mixed. Incubate on ice for 3-5 minutes, centrifuge at 13,000 rpm for 10 minutes, carefully transfer the supernatant. 
Mix immediately after adding Solution P3 to avoid local precipitation of SDS. If there are still tiny white precipitates in the supernatant, centrifuge again and transfer the supernatant.

Pretreat the adsorption column with Equilibration Buffer 
mandatory step; refer to "About the Use of Equilibration Buffer" above for specific methods.
1.Load the supernatant obtained in the previous step into the Spin AC Adsorption Column (placed in a collection tube) in batches. Centrifuge at 13,000 rpm for 30-60 seconds, discard the waste liquid in the collection tube.

2.Optional Step: Add 500 µL of Deproteinization Buffer PE, centrifuge at 13,000 rpm for 30-60 seconds, and discard the waste liquid. 
This step is to remove trace impurities such as nucleases. It is recommended for endA strains (e.g., JM series, HB101) or wild-type strains with high nuclease content; it can be skipped for deficient strains with low nuclease content (e.g., XL-1 Blue, DH5α).

3.Add 600 µL of Wash Buffer WB (check if absolute ethanol has been added!). Centrifuge at 12,000 rpm for 30 seconds and discard the waste liquid.

4.Add another 600 µL of Wash Buffer WB, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid.

5.Place the Spin AC Adsorption Column back into the empty collection tube. Centrifuge at 13,000 rpm for 2 minutes to completely remove residual wash buffer (residual ethanol may inhibit downstream reactions).

6.Transfer the Spin AC Adsorption Column to a clean centrifuge tube. Add 100-250 µL 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 rpm for 1 minute. For larger plasmid yield, reapply the eluate to the spin column and centrifuge for 1 minute.
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 80 µL (excessively small volume reduces plasmid elution efficiency and yield).