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Product Description
MycoSHENTEK® Mycoplasma DNA Extraction Kit (2G) is a magnetic particle–based DNA extraction kit designed for isolation of trace mycoplasma DNA from master cell banks, working cell banks, virus seed banks and complex sample matrices (e.g., up to 10^7 cells culture, 5% human albumin solutions, high-concentration plasmids). The kit is supplied for 50 extractions and is intended to be used in an integrated workflow together with the MycoSHENTEK® Mycoplasma Detection Kit (2G). The method is validated according to USP <63>, EP 2.6.7 and JP XVIII for mycoplasma detection. The kit supports manual extraction and automated extraction using the rHCDpurify system. The validated detection limit is 10 CFU/mL. The protocol includes sample pretreatment, enzymatic digestion (Proteinase K), magnetic-particle binding, wash steps, and elution to produce purified DNA suitable for downstream real-time PCR assays.
Technical Specifications
| Parameter | Details |
|---|---|
| Intended use / scope | Extraction of trace mycoplasma DNA from cell banks, virus seed banks and complex matrices (cell cultures, 5% human albumin, high-concentration plasmids). |
| Validated standards | Validated according to USP <63>, EP 2.6.7 and JP XVIII for mycoplasma detection. |
| Detection limit (LOD) | 10 CFU/mL (validated when used with MycoSHENTEK® Mycoplasma Detection Kit (2G)). |
| Sample input volume | Up to ~400 µL directly; larger volumes should be concentrated by centrifugation and reduced to ~400 µL prior to extraction. |
| Reactions per kit | Reagents sufficient for 50 extractions. |
| Typical digestion/incubation conditions | Add 20 µL Proteinase K and 100 µL Lysis buffer; incubate 55°C for 60 min for most matrices; for 5% human albumin incubate at 25°C for 60 min. |
| Binding step | Per sample add 200 µL Binding Buffer, 200 µL isopropanol and 30 µL Magnetic particles; vertical mixing ~5 min to bind nucleic acids. |
| Wash steps | Two washes: 700 µL Wash buffer A then 700 µL Wash buffer B; remove residual ethanol and air-dry magnetic pellet briefly (30 s–3 min). |
| Elution | Elute with 50 µL Elution buffer; incubate at 70°C for 7 min with intermittent vortexing; recover eluate (~40 µL+ recommended for downstream assay). |
| Automated run time (rHCDpurify) | Approx. 43 minutes per plate run (program: Myco-2G). |
| Storage stability | Kit components stable up to 24 months when stored under recommended conditions (see component list). |
| Critical centrifugation speeds | Debris removal: 7,250×g (10 s); concentration and plasmid/cell concentration steps: 18,000×g (30 min); microcentrifuge brief spins: 3–10 s for liquid collection. |
| Controls | Negative control sample (NCS) procedure provided; Internal Control (IC) (2G) is used during extraction (IC is supplied in the companion detection kit). |
Features
- High analytical sensitivity: Validated detection limit of 10 CFU/mL when used with MycoSHENTEK® Mycoplasma Detection Kit (2G).
- Broad matrix compatibility: Works with cell cultures (up to 10^7 cells), cell culture supernatants, 5% human albumin solutions, high-concentration plasmids and other complex matrices.
- Flexible workflow: Supports both manual magnetic-particle extraction and automated extraction on the rHCDpurify system.
- Pharmacopeial validation: Validated in accordance with USP <63>, EP 2.6.7 and JP XVIII for mycoplasma detection workflows.
- Complete extraction chemistry: Includes lysis, binding, wash and elution reagents plus magnetic particles and proteinase K for robust nucleic acid recovery from low-copy targets.
Applications
- Mycoplasma testing of master cell banks (MCBs) and working cell banks (WCBs)
- Mycoplasma testing of virus seed banks
- Testing of cell culture samples (up to 10^7 cells)
- Testing of cell culture supernatants
- Testing of samples containing 5% human albumin
- Extraction from high-concentration plasmid preparations prior to mycoplasma detection
- Sample preparation prior to real-time PCR-based mycoplasma detection
Kit Contents
Wash buffer A (NND015)Room temperature (after adding 20 mL anhydrous ethanol prior to first use)
Binding solution (NND017)Room temperature
Elution buffer (NND019)Room temperature
Dilution buffer (NND022)Room temperature
Lysis buffer (NND028)Room temperature (heat at 37°C if cloudy)
Pretreatment buffer (NND002) — 1.25 mL × 4 tubesRoom temperature
Magnetic particles (NND033) — 750 µL × 2 tubes2–8 °C
5 M NaCl (NND040) — 500 µL × 1 tubeRoom temperature
Precipitation solution I (NND003) — 25 µL × 1 tubeRoom temperature (dilute 1:99 in DNA Dilution Buffer prior to use)
Precipitation solution II (NND004) — 500 µL × 1 tube-20 °C
Proteinase K (NND023) — 500 µL × 2 tubes-20 °C / according to label (keep frozen until use)
* Total: 50 extractions | Method: Magnetic particle–based DNA extraction (magnetic bead chemistry) for downstream qPCR
Attention
• Read MSDS and use appropriate PPE (eyewear, mask, gloves, lab coat).
• Divide lab into separate areas (negative/preparation, positive/sample prep, amplification) and use dedicated equipment to prevent cross-contamination.
• Ensure ambient temperature is not lower than 22°C before starting experiments (per manual recommendation).
• Centrifuge tubes before opening to avoid splashing; handle tubes carefully to prevent contamination.
• Change gloves frequently and use different gloves/lab coats in separate areas.
• Dispose of used tips and liquid waste according to local regulations; do not open post-PCR products in amplification area.
• Avoid over-drying magnetic particle pellet during ethanol removal to prevent incomplete elution.
• Perform DNA detection the same day as extraction to ensure accuracy.
• For automated runs on rHCDpurify: UV sterilize the instrument for ≥15 minutes and clean inner walls with 75% ethanol wipes before and after runs; minimum interval between extractions should be 30 minutes.
• When removing supernatants, avoid aspirating magnetic particles; use magnetic stand and allow full particle separation (3–5 min) before removing supernatant.
Quality Management & Certifications
Quality System
Download QMSQMS (ISO, GMP)
Download CertificateQuality Advantages
View ReportQuality Control Process
Download ProcessTechnical Resources & Downloads
Frequently Asked Questions (FAQ)
Q1: What is the limit of detection when this extraction kit is used with the MycoSHENTEK® Mycoplasma Detection Kit (2G)?
The workflow is validated with a detection limit of 10 CFU/mL when used with the MycoSHENTEK® Mycoplasma Detection Kit (2G).
Q2: Can I process sample volumes larger than 400 µL?
Yes. For sample volumes >400 µL the protocol requires concentration by centrifugation to a final volume of approximately 400 µL before extraction.
Q3: Which samples require a different digestion temperature?
Most sample types are incubated with Proteinase K at 55°C for 60 minutes. Samples containing 5% human albumin and their controls should be incubated at 25°C for 60 minutes.
Q4: Is the kit compatible with automation?
Yes. The kit is compatible with automated extraction using the rHCDpurify system (example program Myco-2G, ~43 minutes runtime).
Research Use Only
✔
Research Use Only (RUO); validated according to USP <63>, EP 2.6.7 and JP XVIII for mycoplasma detection – This product is intended for laboratory research purposes only and not for clinical or regulatory diagnostic use.
✔
Not intended for use in USDA or FDA regulated diagnostic testing or official compliance testing.
When can I expect my order to ship?
Most orders are filled and shipped within 2-3 business days from the time they are received.
Our standard shipping usually take 2-5 days.
We also provide express shippping for time-sensitive deliveries.
Email contact@biofargo.com if you have any requirements.
