The HEK293 Host Cell Residual DNA Fragment Analysis Kit is designed for the quantitative analysis of HEK293 residual DNA fragments of various lengths in intermediates, semi-finished products, and final products of biological preparations. This kit utilizes the qPCR fluorescence probe principle to specifically and rapidly detect HEK293 DNA residues both below and above 200 base pairs, with a quantification limit as low as 10 fg/μL. It also includes the HEK293 DNA Control (DNA quantitative reference). This kit can be used in conjunction with the company's magnetic bead-based residual DNA sample preparation kits (Cat#18461ES/18462ES).

Product information

Component

Storage and shipping:

1. All components are shipped on dry ice and should be stored at -25°C to -15°C upon receipt. The shelf life is 2 years. Components A and B1, B2, B3, and B4 should be stored protected from light.

2. Upon receipt, verify that all 7 components are present and immediately store them at the recommended temperatures.

Precautions:

1. This product is intended for research purposes only.

2. For safety and health reasons, please wear laboratory coats and disposable gloves during operation.

3. Before using this reagent, carefully read the instruction manual. Experiments should be conducted following standard procedures, including sample handling, preparation of reaction mixtures, and pipetting.

4. Each component should be thoroughly mixed by gentle shaking and briefly centrifuged before use.

Compatible Instruments:

Including but not limited to the following instruments: Thermo Scientific: ABI 7500, ABI Quant Studio 5, ABI Step OnePlus, Bio-Rad: CFX96 Optic Module, Shanghai Hongshi Medical Technology: SLAN-96S

Instructions for Use

 1. Dilution of HEK293 DNA Control Quantitative Reference and Preparation of Standard Curves

 The HEK293 Fragment Analysis Kit includes four amplification fragments of different lengths: 82 bp, 133 bp, 227 bp, and 515 bp. When establishing standard curves, set up separate curves for each amplification fragment and calculate their residual amounts and relative distributions based on the corresponding standard curves.

Use the DNA Dilution Buffer provided in the kit to perform a gradient dilution of the HEK293 DNA Control Quantitative Reference. The dilution concentrations should be: 3 ng/μL, 300 pg/μL, 30 pg/μL, 3 pg/μL, 300 fg/μL, and 30 fg/μL.

The details are as follows

   1). Place the HEK293 DNA Control and DNA Dilution Buffer from the kit on ice to thaw. After complete thawing, gently vortex to mix and centrifuge briefly (10 seconds) to collect the solution at the bottom of the tube.

   2). Prepare six clean 1.5 mL centrifuge tubes and label them as Std0, Std1, Std2, Std3, Std4, and Std5.

   3). In the tube labeled Std0, add 90 μL of DNA Dilution Buffer and 10 μL of HEK293 DNA Control to achieve a concentration of 3 ng/μL. Vortex gently to mix and centrifuge briefly (10 seconds). This concentration can be aliquoted and stored at -20°C for short-term use (up to 3 months). Avoid repeated freeze-thaw cycles.

   4). In the tubes labeled Std0, Std1, Std2, Std3, Std4, and Std5, first add 90 μL of DNA Dilution Buffer to each. Each dilution step should be mixed gently and centrifuged briefly to ensure uniformity. Then perform gradient dilutions as follows:    

Table 1: Standard Gradient Dilution

* For each concentration, perform 3 replicates. This reagent can test within a linear range of 300 pg/μL to 30 fg/μL. If needed, the linear range can be appropriately extended or narrowed.

** To reduce repeated freeze-thaw cycles and avoid contamination, it is recommended to aliquot and store the DNA quantification standard at -20°C for the first use.

*** Unused, thawed DNA dilution can be stored at 2-8°C for up to 7 days. If not used for an extended period, store it at -20°C.

**** To ensure complete mixing of the template, gently shake each gradient dilution for about 1 minute.

2. Preparation of Test Sample (TS)

Prepare the test sample TS according to the experiment setup, as follows:  

1) Take 100 μL of the test sample and add it to a 1.5 mL clean centrifuge tube. Label it as TS, perform sample pre-treatment, and prepare to purify the test sample.  

2) To meet the requirement for analyzing four different extension lengths simultaneously, the amount of pre-treated test sample should be ≥120 μL. Therefore, it is recommended to prepare 2 tubes of each sample for pre-treatment, and after extraction, mix them together for use.

3. Preparation of Negative Extraction Control (NCS)

Prepare the negative extraction control NCS according to the experiment setup, as follows:  

1) Take 100 μL of the sample matrix solution (or DNA dilution buffer) and add it to a 1.5 mL clean centrifuge tube. Label it as NCS.  

2) Perform the sample pre-treatment of the negative control NCS together with the batch of test samples and prepare the purified negative control NCS solution.  

3) To meet the requirement for analyzing four different extension lengths simultaneously, the amount of pre-treated NCS sample should be ≥120 μL. Therefore, it is recommended to prepare 2 tubes of each NCS sample for pre-treatment, and after extraction, mix them together for use.

4. Preparation of No Template Control (NTC)

Prepare the no template control NTC according to the experiment setup, as follows:  

1) No Template Control (NTC) does not require sample pre-treatment, and can be prepared starting from the stage of detecting residual DNA content using qPCR.  

2) For each tube or well, the NTC sample consists of 20 μL of mix (i.e., 15 μL HEK293 qPCR Mix + 5 μL corresponding HEK293 Primer & Probe Mix) + 10 μL DNA Dilution Buffer. It is recommended to prepare enough for 3 replicate wells.

5. qPCR reaction system

Table 2. Reaction system for 82 bp fragment

Table 3. Reaction system for 133 bp fragment

Table 4. Reaction system for 227 bp fragment

Table 5. Reaction system for 515 bp fragment

* To calculate the total amount of Mix required for this reaction based on the number of wells:  

Mix = (Number of reaction wells + 2) × (15 + 5) μL (to account for the 2 wells' loss). Typically, 3 replicate wells are prepared for each sample.

** Number of reaction wells = (5 concentration gradient standard curve wells + 1 No Template Control (NTC) + 1 Negative Control Solution (NCS) + N Test Samples (TS)) × 3.

NTC (No Template Control): DNA Dilution Buffer  

NCS (Negative Control Solution): Sample matrix solution or DNA Dilution Buffer after sample pre-treatment to obtain the purified solution, which is the NCS.  

TS (Test Sample): The sample to be tested.

***After dispensing the samples and sealing the tubes, briefly centrifuge at low speed (10 sec) to collect the liquid from the tube walls to the bottom. Then vortex for at least 5 seconds to mix thoroughly. Afterward, perform another low-speed centrifugation (10 sec) . If there are any bubbles, make sure to remove them.

Table 6: Reference Plate Layout

This example shows the qPCR detection procedure for analyzing the amplification fragments of residual HEK293 DNA. The test samples include: 5 concentration gradients of HEK293 DNA standard curve, 1 test sample (TS), 1 negative control solution (NCS), and 1 no-template control (NTC). It is recommended to run 3 replicate wells for each sample.

6. Amplification Program Parameters (Three-step Method) (Example using ABI 7500 qPCR Instrument, Software Version 2.0)**

1) Create a new blank program and select "Absolute Quantification" as the detection template.

2) For the four different amplification fragment lengths, create new detection probes, naming them "HEK293-82", "HEK293-133", "HEK293-227", and "HEK293-515". Select the reporter fluorophore as "FAM" and the quencher fluorophore as "none". Set the reference dye for detection as "ROX" (the reference dye may be added or not depending on the instrument model and other factors).

3) In the "Assign target(s) to the selected wells" panel, set the "Task" field for the standard curve wells as "Standard", and assign the corresponding values in the "Quantity" field as "300000", "30000", "3000", "300", "30" (representing the DNA concentration per well, in fg/μL). Name the wells in the "Sample Name" field as "300 pg/μL", "30 pg/μL", "3 pg/μL", "300 fg/μL", "30 fg/μL". For the NTC wells, set the "Task" to "NTC". For the NCS and TS wells, set the "Task" to "Unknown", and name the wells "NCS" and "TS" in the "Sample Name" field. After setting these parameters, click "Start Run" to begin the instrument run.

4) Amplification program settings: Set the three-step amplification program, with a reaction volume of 30 μL.

Table7. PCR procedure

7. qPCR Result Analysis

1) In the "Analysis" panel under "Amplification Plot," the system will automatically set the "Threshold." Sometimes, the default "Threshold" is too close to the baseline, causing significant Ct variation between replicates. You can manually adjust the "Threshold" to an appropriate position and click "Analyze." At this point, you can preliminarily check the amplification curves in the "Multicomponent Plot" to see if they are normal.  

2) In the "Analysis" panel under "Standard Curve," you can read the standard curve's R², amplification efficiency (Eff%), slope, and intercept. For a normal standard curve: R² > 0.99, amplification efficiency (90% ≤ Eff% ≤ 110%), and slope between -3.6 and -3.1.  

3) In the "Analysis" panel under "View well table," you can read the "Quantity" column for no-template control (NTC), negative control (NCS), and test samples (TS), with the unit in fg/μL. The units can be converted later in the report.  

4) The parameter settings for result analysis should depend on the specific instrument model and software version. Usually, the instrument can automatically interpret the results.  

5) The Ct value of the negative control (NCS) should be greater than the average Ct value of the standard curve's lowest concentration.  

6) The result of the no-template control (NTC) should be "Undetermined" or have a Ct value ≥ 32.

Document

Manual