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Jianshi Biotechnology (Beijing) Co., Ltd.

Endotoxin Removal Plasmid Mini‑to‑Mid Prep Kit (PLUS) (TD435-PLUS)

Item number	Packaging Specifications	Unit	Price (CNY)
TD435-PLUS-10	10 times	box	100
TD435-PLUS-50	50 times	box	350
TD435-PLUS-200	200 times	box	1400

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GENSTONE BIOTECH’s Endotoxin‑Depleted Plasmid Mini‑Prep Mid‑Volume Kit (PLUS Version, Product Catalog No.: TD435‑PLUS Series) is an upgraded purification solution specifically designed to isolate ultra‑low‑endotoxin, high‑purity plasmid DNA, making it particularly well suited for sensitive downstream applications that demand stringent nucleic acid purity.

Building on the efficient extraction of plasmid DNA, this kit’s core advantage lies in its integration of a unique… Colorimetric reaction With professionalism Endotoxin Removal Protocol During the procedure, changes in solution color—such as the magenta of P1, the pale pink after lysis, and the yellow after neutralization—provide an immediate visual indication of the experimental status, greatly enhancing both intuitiveness and controllability. When used in conjunction with a dedicated endotoxin‑removal reagent and an optimized purification protocol, this method can efficiently adsorb and eliminate endotoxins from the sample.

The key features of this kit include:

Ultra-low endotoxin residue : Utilizing a proprietary endotoxin removal technology, the purified plasmid DNA can achieve an endotoxin level as low as ≤0.05 EU/μg , achieving “ultra‑low residue” levels and even “endotoxin‑free” standards (≤0.025 EU/μg), thereby meeting the demanding requirements of cutting‑edge applications such as gene therapy, AAV production, CAR‑T research, and transfection of highly sensitive cell lines.

Intuitive and safe operation : The colorimetric reaction provides clear, real-time indication, eliminating the need for complex interpretation. The procedure does not require toxic reagents such as phenol or chloroform, nor does it involve ethanol precipitation, making it both safe and convenient.

High purity and high yield : The extracted plasmid exhibits excellent purity, with an A260/A280 ratio ≥ 1.8 and an A260/A230 ratio ≥ 2.0; the yield per extraction can reach Above 200 μg The plasmid recovery rate is between 60% and 80% (after the endotoxin removal step).

Fast and flexible : The basic extraction process (Part 1) takes only about 20–30 minutes (using vacuum or centrifugation). For maximum purity, an optional second step—deep endotoxin removal—can be performed, bringing the total time to approximately 45 minutes. It also supports Centrifugal and Negative pressure Two purification modes, tailored to different throughput levels and equipment configurations.

Wide compatibility ： Can efficiently extract large plasmids up to 25 kb in size. , suitable for both high-copy and low-copy plasmids.

Main components The kit includes RNase A solution, resuspension buffer P1 (red), lysis buffer P2, neutralization buffer P4, endotoxin removal solution, plasmid DNA pre‑wash solution, Endo‑Free plasmid DNA wash buffer, DNase/RNase‑free water, purification column No. 3, a 2 mL collection tube, and a 25 mL funnel (for vacuum‑assisted operations).

Downstream applications : The ultra‑low endotoxin, high‑purity plasmid DNA prepared with this kit can be used directly in experiments that demand exceptionally high nucleic acid quality, such as Cell transfection (especially in primary cells, stem cells, and immune cells), in vitro transcription, viral packaging (e.g., AAV), in vivo experiments, and gene therapy research. as well as standard procedures such as restriction enzyme digestion, cloning, sequencing, and PCR.

Core Advantages

Ultra-low endotoxin : Endotoxin content ≤0.05 EU/μg
Colorimetric indication : P1 Red → Cleavage to pale pink → Neutralization turns yellow, for intuitive monitoring
Operational Safety : No toxic reagents such as phenol/chloroform are required.
Flexible and efficient : Supports the vacuum method (~20 min) and the centrifugation method (~30 min)

Technical Specifications

Endotoxin levels : Conventional extraction 0.05–1 EU/μg
Purity index :A₂₆₀/A₂₈₀≥1.8, A₂₆₀/A₂₃₀≥2.0
Recommended OD : OD₆₀₀ = 2–3, incubate overnight for 14 h
Plasmid yield : Recovery rate after advanced treatment 60%-80%

Key Notes

P1 Preprocessing : RNase A must be added to a final concentration of approximately 100 μg/ml.
Detergent Preparation : Anhydrous ethanol must be added upon first use.
Bacterial Suspension Control : Avoid excessive bacterial lysate to prevent residual genomic DNA.
Storage conditions : Store at different temperatures according to component type

TD435-PLUS Endotoxin‑Free Plasmid Mini‑Prep Kit – High‑Throughput FAQ (10 Highly Valuable Questions)

1. What is the endotoxin removal level of this kit, and which highly sensitive experimental requirements can it meet?

This product employs a proprietary combination of endotoxin-removal solution and column-based purification, reducing endotoxin levels to as low as ≤0.05 EU/μg , conventional extraction can achieve 0.05–1 EU/μg,

Meets the requirements of highly sensitive applications such as transfection‑grade reagents, AAV production, CAR‑T cell engineering, primary and stem cell transfection, and preclinical research.

The first step alone is sufficient for routine cell transfection, while the second, more intensive processing step can achieve ultra‑low residual levels and meet endotoxin‑free standards.

2. What is the purpose of the color development system in the kit? What issues do abnormal colors indicate, respectively?

The unique P1 red → cleavage to pale pink → neutralization to yellow coloration allows for intuitive assessment of the experimental status:

The cells have not been adequately resuspended: Uneven red color and clumping → insufficient pyrolysis, leading to reduced yield.
Incomplete cracking: Pale pink, turbid, and non-viscous → residual genomic DNA, reduced purity
Incomplete neutralization: Red coloration not fully removed → poor removal of protein/endotoxins, which may inhibit downstream assays.

No additional checks are required throughout the process, significantly reducing the error rate for novice users.

3. Does the extraction require phenol/chloroform or ethanol precipitation? What are the key advantages of this method compared to conventional approaches?

Absolutely not needed Toxic reagents such as phenol and chloroform also eliminate the need for an isopropanol/ethanol precipitation step:

Safety: No risk of toxicity or residue from organic reagents.

Efficient: The negative-pressure method takes about 20 minutes, while the centrifugation method takes about 30 minutes to complete.

High purity: Free from organic solvent interference, the plasmid can be directly used for restriction digestion, sequencing, and transfection.

Stability: Avoid losses due to precipitation, and enhance recovery rates and batch-to-batch consistency.

4. How do you choose between vacuum operation and centrifugal operation? What are the key differences between these two methods?

Method	Time-consuming	Applicable Scenarios	Key Takeaways
Negative pressure method	~20 minutes	High-throughput, multiplexed parallel processing	Requires a vacuum manifold; liquid filtration is faster, with less residue on the membrane.
Centrifugation	~30 minutes	Single-sample, non-negative-pressure equipment	Each loading should not exceed 800 μL; repeat the loading until the entire mixture has been filtered.

Both methods yield consistent results in endotoxin removal, recovery rate, and purity, allowing flexible selection based on laboratory equipment.

5. What are the root causes of low plasmid yield, excessive endotoxin levels, and high residual RNA, and what are the corresponding solutions?

Low yield: OD of the bacterial suspension > 3, cells not resuspended, P2 precipitation at low temperature, aspiration of the pellet, and insufficient elution → maintain OD at 2–3, resuspend P2 at 37°C, avoid aspirating the pellet, and preheat the elution buffer.

Endotoxin levels exceed the limit: Excessive microbial load, endotoxin‑contaminated consumables, and insufficient mixing of the endotoxin removal solution → strictly control the sample loading volume, use endotoxin‑free consumables throughout the process, and ensure thorough shaking or 360° inversion for complete mixing.

RNA Residue: P1 without RNase A, with excessive bacterial load → P1 must include RNase A, be stored at 4°C, and reduce the bacterial suspension volume.

6. What are the key considerations in preparing reagents? What experimental failures can result from improper preparation?

P1: RNase A must be added to a final concentration of approximately 100 μg/ml and stored at 4°C; omission or inactivation will result in significant RNA contamination and artificially elevated concentrations.

Endo-Free Washing Solution: For the first use, add anhydrous ethanol in a ratio of 15 mL + 60 mL; or 60 mL + 240 mL. If no ethanol is added, the plasmid will not bind to the column, and extraction will fail.

Endotoxin removal solution: Store at 4°C; use after shaking to ensure uniformity—no impact on efficacy.

7. How should the OD of the bacterial suspension and the loading volume be controlled? What irreversible harms can result from an excessive amount of bacterial suspension?

Recommendation OD₆₀₀=2–3 , an overnight incubation of approximately 14 hours is optimal; adjust the sample volume according to the OD (e.g., use 15 mL at OD = 2, and 3 mL at OD = 10).

Harm caused by excessive bacterial suspension:

Incomplete lysis and neutralization result in substantial residual genomic DNA and proteins.
RNA overload, RNase A cannot fully degrade it.
Endotoxin levels surge, far exceeding the removal capacity.
The purification column is overloaded, resulting in a simultaneous decline in both yield and purity.

8. Is the second-step deep endotoxin removal step mandatory? What impact does it have on plasmid recovery yield?

Conventional cell transfection: Only the first part is required; endotoxin ≤ 1 EU/μg meets the requirement.

Primary cells, stem cells, AAV/CAR-T, in vivo experiments: Part two is mandatory; endotoxin levels can be reduced to ≤0.05 EU/μg

Recovery rate: After deep processing, a plasmid recovery rate of 60%–80% and a concentration reduction of 10%–30% are considered normal; following RNA and impurity removal, the measured purity is higher and downstream performance is more stable.

9. What are the purity metrics of the isolated plasmid? Which downstream applications can it be directly used for?

Purity meets the standard: A₂₆₀/A₂₈₀ ≥ 1.8, A₂₆₀/A₂₃₀ ≥ 2.0, free of protein, salts, endotoxins, RNA, and residual genomic DNA.

Can be used directly for: High-sensitivity assays such as restriction digestion, transformation, PCR, in vitro transcription, Sanger and high-throughput sequencing, cell transfection, in vitro genome editing, and preclinical studies require no secondary purification.

10. What are the storage conditions for the kit components? If turbidity or precipitation occurs, can the kit still be used normally?

Store at 4°C: RNase A, P1, endotoxin removal solution, plasmid DNA pre‑wash solution

Store at room temperature: P2, P4, Endo-Free Wash Buffer, Enzyme-Free Water, Purification Columns, Collection Tubes, Funnel

Exception Handling:

P2 Low-Temperature Turbidity: Dissolve in a 37°C water bath; do not shake vigorously to prevent foaming.
Turbidity of the endotoxin removal solution: 4°C is normal; simply mix thoroughly before use.

Tip: All reagents should be protected from high temperatures, strong light, and prolonged exposure to open air to prevent degradation.

Endotoxin Removal Plasmid Mini‑to‑Mid Prep Kit (PLUS) (TD435-PLUS)

TD435-PLUS Endotoxin-Free Plasmid Mini‑Prep Kit, Mid‑Volume Format

Core Advantages

Technical Specifications

Key Notes

TD435-PLUS Endotoxin‑Free Plasmid Mini‑Prep Kit – High‑Throughput FAQ (10 Highly Valuable Questions)