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Product Description
SHENTEK® Residual E1A DNA Quantitation Kit is designed for the quantitation of residual E1A DNA derived from host cells (e.g., HEK293 and 293T) across different stages of biopharmaceutical manufacture, from in-process samples to final products. The kit uses fluorescent quantitative PCR (real-time qPCR) for rapid, specific and reliable absolute quantitation. An Internal Positive Control (IPC) is included to monitor PCR performance and potential inhibition. The kit provides both E1A linear DNA Control and E1A non-linear DNA Control to accommodate different user needs. Recommended sample extraction procedures are provided in the SHENTEK® Residual Host Cell DNA Sample Preparation Kit User Guide (Product No. 1104191).
Technical Specifications
| Parameter | Details |
|---|---|
| Intended reactions | Reagents for 100 reactions |
| Analytical standard range (linear DNA control) | 4.97 × 10^8 copies/μL (stock) with serial 10-fold dilutions down to 4.97 × 10^1 copies/μL (ST1–ST6 covering 4.97 × 10^6 to 4.97 × 10^1 in standard curve steps) |
| Analytical standard range (non-linear DNA control) | 2.98 × 10^7 copies/μL (stock) with serial 10-fold dilutions down to 2.98 × 10^2 copies/μL (ST0–ST5 covering 2.98 × 10^6 to 2.98 × 10^2 in standard curve steps) |
| Reaction volume | 30 μL total reaction volume (20 μL qPCR MIX + 10 μL sample or control) |
| qPCR MIX composition (per reaction) | qPCR Reaction Buffer 15.9 μL; E1A Primer & Probe MIX 2.8 μL; IPC MIX 1.3 μL (total 20 μL qPCR MIX) |
| Sample input | 10 μL purified test sample or purified NCS per reaction; recommended starting material for extraction: 100 μL sample |
| Thermal cycling conditions | Activation: 95 °C 10:00 (1 cycle); Denaturation: 95 °C 00:15 (40 cycles); Annealing/Extension: 60 °C 01:00 (fluorescence read during this step) |
| Fluorescence detection dyes | E1A-DNA detector: CY5; IPC detector: VIC; Passive reference: ROX |
| Analysis settings / thresholds | Manual Ct with Threshold = 0.02 for both E1A-DNA and IPC; Automatic baseline recommended |
| NTC acceptance criteria | Ct value of NTC should be ≥ 35.00 cycles or undetermined |
| IPC acceptance criteria | Ct(IPC) of sample should be within ±1.0 Ct of Ct(IPC) of NCS; significant shift indicates possible inhibition |
| LOQ / LOD | Limit of quantification (LOQ) not specified in kit documentation; users should determine proven LOQ during method validation. If proven LOQ is lower than lowest standard, Ct(NCS) should be larger than Ct(LOQ). |
| Recommended replicates and standard points | Triplicate measurements recommended; at least five concentration points recommended for standard curve (six when using linear DNA control) |
| Validated instruments | SHENTEK-96S, ABI 7500 (SDS v1.4 example), Bio-Rad CFX96 (other compatible real-time PCR systems may be used) |
| Storage stability | -20 °C (stable for 24 months) |
Features
- Specific and sensitive absolute quantitation: Fluorescent qPCR assay for quantitative detection of residual E1A DNA with serial standards to create an absolute standard curve.
- Internal Positive Control (IPC): IPC included to monitor PCR performance and identify potential sample inhibition; IPC results used to assess recovery when spiked samples are tested.
- Two control formats provided: Both E1A linear DNA Control (lyophilized) and E1A non-linear DNA Control are supplied so users can choose according to validation needs.
- Ready-to-use mixes: qPCR Reaction Buffer, Primer & Probe MIX and IPC MIX supplied in ready-to-use formats to simplify setup; protect light-sensitive reagents from light.
- Compatibility: Validated on common real-time PCR platforms (SHENTEK-96S, ABI 7500, CFX96); thermal and detector settings provided for instrument configuration.
Applications
- Quantitation of residual E1A DNA from host cells (e.g., HEK293, 293T) in biopharmaceutical development and manufacturing
- In-process sample testing and final product testing for residual host-cell E1A DNA
- Method validation and QC workflows requiring absolute quantitation of E1A sequences
Kit Contents
E1A linear DNA Control (lyophilized powder × 1 tube)-20 °C
E1A non-linear DNA Control (50 μL × 1 tube)-20 °C
qPCR Reaction Buffer (850 μL × 2 tubes)-20 °C, protect from light
E1A Primer & Probe MIX (300 μL × 1 tube)-20 °C, protect from light
IPC MIX (150 μL × 1 tube)-20 °C, protect from light
DNA Dilution Buffer (DDB) (1.5 mL × 3 tubes)-20 °C
* Total: 100 reactions | Method: qPCR
Attention
• For Research Use Only (RUO). Not for diagnostic use.
• Read Material Safety Data Sheets (MSDS) and follow laboratory safety procedures; wear appropriate PPE (gloves, eyewear, mask, lab coat).
• Prevent nucleic acid contamination: irradiate work surfaces, pipettes and tubes with UV for 30 minutes and disinfect with 75% ethanol before setup.
• Thaw reagents at 2–8 °C or on ice; vortex and briefly centrifuge before use. Protect light-sensitive reagents from light.
• Store kit components at -20 °C and check expiration dates. Do not use if reagents show precipitate or cloudiness (heat DDB at 37 °C to clarify if necessary).
• Include appropriate controls: NTC, NCS, standards and IPC. Use triplicates for samples and standards for reliable quantitation.
• Instrument-specific settings should be adapted to the user's instrument and software version; follow provided detector/dye assignments and thermal profile as guidance.
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 sample types can be tested with this kit?
The kit is intended for quantitation of residual E1A DNA from host cell-derived samples (for example, HEK293 and 293T) and is applicable to samples collected during biopharmaceutical manufacturing (in-process and final product). Samples must be purified prior to qPCR according to the recommended extraction workflow.
Q2: How is the standard curve prepared and what dynamic range is covered?
Standards are prepared by serial 10-fold dilutions of the provided DNA controls. The linear DNA control stock is 4.97 × 10^8 copies/μL (diluted down to 4.97 × 10^1 copies/μL across ST1–ST6); the non-linear control stock is 2.98 × 10^7 copies/μL (diluted down to 2.98 × 10^2 copies/μL across ST0–ST5). At least five standard points (six for linear control) are recommended.
Q3: What quality criteria should I use to accept a run?
NTC should be undetermined or have Ct ≥ 35. IPC Ct for sample should be within ±1.0 Ct of IPC Ct for NCS; significant shifts indicate inhibition. Verify standard curve slope, intercept and R^2 before reporting sample quantities.
Q4: Is the limit of quantification (LOQ) provided?
A numeric LOQ is not specified in the kit documentation. Users should determine the proven LOQ during method validation for their specific matrix and instrument. If proven LOQ is below the lowest standard, Ct(NCS) should be larger than Ct(LOQ).
Research Use Only
✔
Research Use Only (RUO) – 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.
