Products

Endo-Free HighPure Plasmid Extraction Maxi Kit(SpinColumn)

Packing Specification：

Product Introduction:

This kit uses an improved SDS-alkaline lysis method to lyse cells. Endotoxin in the crude extract 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 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.Eliminates the need for toxic reagents such as phenol and chloroform, as well as ethanol precipitation. Rapid and convenient: 0.2-1.5 mg of pure high-copy plasmid DNA can be quickly extracted from 150-300 mL of E. coli LB (Luria-Bertani) culture, with an extraction rate of 80-90%.
3.Unique 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 0.2-1.5 mg of pure, endotoxin-free high-copy plasmid DNA from 150-300 mL of E. coli LB culture.

Operation Steps:
Tips:
Before first use, add 100 mL of absolute ethanol to each of the two Wash Buffer WB bottles, 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.Add 150-200 mL (maximum 300 mL) of overnight bacterial culture to a 50 mL centrifuge tube, centrifuge at 8,000 x g (approximately 9,000 rpm) for 1 minute, 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 6-8 times to fully lyse the bacteria, and incubate at room temperature for 4-5 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 Solution N3, immediately invert up and down gently 6-8 times. White flocculent precipitation will form when fully mixed. Centrifuge at 8,000 x g (approximately 9,000 rpm) for 10-15 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.

5.Add 0.1 volumes of Endotoxin Remover (10% of the supernatant volume, approximately 2.4 mL) 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 8,000-10,000 x g 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.

8.Add 0.5 volumes of isopropanol (approximately 11 mL) to the upper aqueous phase, invert thoroughly to mix. Load the mixture into the Spin DC Adsorption Column (placed in a collection tube) in multiple batches (≤15 mL per aliquot). Centrifuge at 8,000 x g (approximately 9,000 rpm) for 1 minute, discard the waste liquid in the collection tube. Repeat until all the mixture has passed through the adsorption column.

9.Add 10 mL of Deproteinization Buffer PE (check if absolute ethanol has been added!). Centrifuge at 8,000 x g (approximately 9,000 rpm) for 1 minute and discard the waste liquid.

10.Add 10 mL of Wash Buffer WB (check if absolute ethanol has been added!). Centrifuge at 8,000 x g (approximately 9,000 rpm) for 1 minute and discard the waste liquid. Add another 10 mL of Wash Buffer WB and repeat the washing step.

11.Place the Spin DC Adsorption Column back into the empty collection tube. Centrifuge at maximum speed (preferably >12,000 x g, approximately 10,000 rpm) for 3 minutes to dry residual ethanol on the matrix membrane. Aspirate any residual ethanol between the inner ring pressure ring and the column wall with a pipette tip.
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.

12.Transfer the Spin DC Adsorption Column to a clean centrifuge tube, open the cap and air-dry at room temperature for 2-3 minutes. 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 3 minutes, then centrifuge at 12,000 x g (approximately 10,000 rpm) for 3 minutes.
Recommendation: To increase plasmid recovery efficiency, reapply the eluate to the spin column, incubate at room temperature for 3 minutes, and centrifuge at 12,000 x g (approximately 10,000 rpm) for 3 minutes. Eluting twice can increase the concentration by approximately 10%.
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).