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Endo-Free Plasmid Extraction Midi Kit(SpinColumn)

Packing Specification：

Product Introduction:
This kit uses an improved SDS-alkaline lysis method to lyse cells. Endotoxin is selectively bound and removed by centrifugation using a unique Endotoxin Remover. The silica matrix membrane in the spin column then 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 high-quality specially manufactured adsorption membranes, ensuring minimal variation in adsorption capacity between columns.
2.Unique process formulation for endotoxin removal, resulting in extremely low endotoxin content (<0.1 EU/μg DNA) and excellent cell transfection efficiency. The plasmid can also 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 15-70 µg of pure, endotoxin-free high-copy plasmid DNA from 5-15 mL of E. coli LB culture.

Operation Steps: 
Tips:
Before first use, add the specified volume of absolute ethanol to the Wash Buffer WB 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 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 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 500 µL of Solution N3, immediately invert up and down gently 6-8 times. White flocculent precipitation will form when fully mixed. Centrifuge at 13,000 rpm for 10 minutes, carefully transfer the supernatant to a new tube, and avoid aspirating the floating white precipitate.

5.Add 0.1 volumes of Endotoxin Remover (10% of the supernatant volume, approximately 160 µL) to the supernatant obtained in the previous step. Invert and rotate to mix, then place on ice (or in crushed ice/refrigerator freezer) for 5 minutes until the turbidity clears (or remains slightly turbid), mixing occasionally.
The supernatant will become turbid after adding Endotoxin Remover, but should clear (or remain slightly turbid) after ice incubation.

6.Incubate at room temperature for 3-5 minutes. The solution will quickly become turbid as it returns to room temperature; invert to mix.
If the room temperature is low or to speed up the process, incubate in a 37-42℃ water bath to accelerate turbidity formation, then invert to mix.

7.Centrifuge at 14,000 x g (12,000 rpm) at room temperature for 10 minutes to separate phases. The upper aqueous phase contains DNA, and the lower blue oily phase contains endotoxins and other impurities. Transfer the upper aqueous phase containing DNA to a new tube (being careful not to aspirate the blue oily layer, which contains endotoxins and other impurities) and discard the oily layer.

Pretreat the adsorption column with Equilibration Buffer
mandatory step; refer to "About the Use of Equilibration Buffer" above for specific methods.
8.Add 0.5 volumes of isopropanol (approximately 740 µL) to the upper aqueous phase, invert thoroughly to mix. Load the mixture into the Spin AC Adsorption Column (placed in a collection tube) in multiple batches (≤700 µL per aliquot). Centrifuge at 12,000 x g for 1 minute, discard the waste liquid in the collection tube. Repeat until all the mixture has passed through the adsorption column.

9.Add 500 μL of Deproteinization Buffer PE (check if absolute ethanol has been added!). Centrifuge at 12,000 rpm for 30 seconds and discard the filtrate.

10.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.

11.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).

12.Transfer the Spin AC Adsorption Column to a clean centrifuge tube. Add 100-200 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 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 100 µL (excessively small volume reduces plasmid elution efficiency and yield).