Products

HighYield Plasmid Extraction Mini Kit(SpinColumn)

Packing Specification：

Product Introduction:
This kit uses a unique high-yield SDS-alkaline lysis formulation to lyse cells, increasing plasmid yield by 1-2 times. 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.Specially improved high-yield buffer formulation increases plasmid yield by 1-2 times.
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:
This kit is suitable for nuclease-deficient strains with low nuclease content, such as XL-1 Blue, Top10, and DH5α. It can also efficiently process strains with high nuclease content, such as JM series and HB101.

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.

1.Take 1.5-5 mL of overnight bacterial culture, centrifuge at 12,000 rpm for 30 seconds, pour off the supernatant as much as possible, and collect the bacterial pellet. 
For collecting more than 1.5 mL of bacterial culture: Centrifuge to discard the supernatant, add more bacterial culture to the same 1.5 mL tube, and repeat Step 1 until sufficient bacterial pellet is collected.

2.Resuspend the bacterial pellet with 250 µL of Solution P1, vortex until completely suspended. 
Undissolved bacterial clumps will affect lysis, leading to low yield and purity.

3.Add 250 µL of Solution P2, gently invert up and down 6-8 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 250 µL of Solution N3, immediately invert up and down gently 6-8 times. White flocculent precipitation will form when fully mixed. Centrifuge at 12,000 rpm for 10 minutes, carefully transfer the supernatant to a new tube, and avoid aspirating the floating white precipitate.
Mix immediately after adding Solution N3 to avoid local precipitation of SDS.

Pretreat the adsorption column with Equilibration Buffer
mandatory step; refer to "About the Use of Equilibration Buffer" above for specific methods.
5.Add 0.5 volumes of isopropanol (approximately 335 µL) to the supernatant, invert thoroughly to mix. Load the mixture into the Spin AC Adsorption Column (placed in a collection tube) in two batches (≤700 µL per aliquot). Centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid in the collection tube. Repeat until all the mixture has passed through the adsorption column.

6.Optional Step: Add 500 µL of Deproteinization Buffer PE, centrifuge at 12,000 rpm for 30 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, Top10, DH5α).

7.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. Add another 600 µL of Wash Buffer WB and repeat the washing step.

8.Place the Spin AC Adsorption Column back into the empty collection tube. Centrifuge at 12,000 rpm for 2 minutes to completely remove residual wash buffer (residual ethanol may inhibit downstream reactions).

9.Transfer the Spin AC Adsorption Column to a clean centrifuge tube. Add 50-100 µL of Elution Buffer EB to the center of the adsorption membrane. Incubate at room temperature for 2 minutes, then centrifuge at 12,000 rpm for 1 minute. For larger plasmid yield, reapply the eluate to the spin column, incubate at room temperature for 2 minutes, 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 30 µL (excessively small volume reduces plasmid elution efficiency and yield).