E. coli Residual DNA Size Analysis Kit-HYI162

Components & Storage

The kit components can be stored at appropriate conditions for up to 24 months. Please check the expiration date on the labels.

Applied instruments, including but not limited to the following

• 7500 Real-Time PCR System
• CFX96 Real-Time PCR System
• Lightcycler480 Real-Time PCR System

Required materials not included in the kit

• Nonstick, DNase-free&Low Retention Microfuge Tubes, 1.5mL
• Nonstick, Low Retention Tips, 1000μL, 100μL, 10μL
• 96-well qPCR plates or PCR 8-strip tubes

Related equipment

• Real-Time PCR System
• Vortex mixer
• Micro plate shaker
• Pipettes, 1000μL, 100μL, 10μL
• Microplate shaker

Workflow

Instructions

1, Experiment preparation

1.1 Wear appropriate protective eyewear, mask, clothing and gloves.

1.2 Irradiate the tabletop, pipettes and tubes with UV for 30 minutes, and disinfect with 75% ethanol.

1.3 Thaw the kit completely at 2-8°C or melt on ice.

2, DNA Control serial dilutions for the standard curve

Note: The kit contains four E.coli primer & probe mixes for different fragment lengths. Please set up four separate standard curves corresponding to each fragment length.

Please check the concentration on the label of the tube containing the E.coli DNA Control prior to dilution.

Prepare four sets of E.coli DNA Control solution with DNA Dilution Buffer (DDB) following the serial dilution procedure below:

2.1 Thaw E.coli DNA Control and DDB completely at 2-8°C or melt on ice. Vortex to mix well and quickly spin down the reagents for 3-5 seconds in microcentrifuge, and repeat 3 times.

2.2 Label six nonstick 1.5 mL microfuge tubes: ST0, ST1, ST2, ST3, ST4 and ST5.

2.3 Dilute the DNA Control to 3000 pg/μL with DDB in the ST0 tube. Vortex to mix well and quickly spin down the reagents for 3-5 seconds in microcentrifuge, and repeat 3 times to mix thoroughly.

2.4 Add 180 μL DDB to each tube of ST1, ST2, ST3, ST4 and ST5.

2.5 Perform the serial dilutions according to Table 2:

Table 2. Dilution for E.coli DNA Control

• The remaining unused DDB need to be stored at 2-8℃. If the solution is cloudy or contains precipitates, heat at 37℃ until it clears.
• At least five concentration of standard curve should be included. To select appropriate sample dilutions, we recommend performing method validation before sample testing.

3, Sample preparation

Add 100 μL of DDB to a new 1.5 mL microfuge tube, and label as NCS. NCS and samples should be prepared in same way for DNA extraction.

4, qPCR MIX preparation

4.1 Determine the number of reaction wells based on the standard curve, with the number of test samples and control samples. Generally, triplicates are tested for each sample. Number of reaction wells = (standard curve of 5 concentration gradients + 1 NTC + 1 NCS + test samples)×3

4.2 Prepare qPCR MIX seperately according to Table 3 - 6.

Table 3. qPCR MIX-85 preparation

Table 4. qPCR MIX-103 Preparation

Table 5. qPCR MIX-220 Preparation

Table 6. qPCR MIX-550 Preparation

For simultaneous detection of the four fragments, please prepare at least 120 μLtemplate DNA for four assays. We recommend to prepare 2 tubes of each sample for pre-treatment at the same time and pool them after extraction.

4.3 After thoroughly mixing qPCR MIX, follow 20 μL each tube is divided into PCR 8-strip tubes or 96-well qPCR plate.

5, qPCR Reaction MIX preparation

5.1 Prepare qPCR Reaction MIX according to Table 7-10 and 96-well plate layout as shown in Table 11.

Table 7. qPCR Reaction MIX-85 Preparation

Table 8. qPCR Reaction MIX-103 Preparation

Table 9. qPCR Reaction MIX-220 Preparation

Table 10. qPCR Reaction MIX-550 Preparation

Table 11. Example of 96-well Plate layout

• This example represents four assays, including selected standard curve points of E.coli DNA Control (ST1-ST5),1 NTC,1 NCS and 1 test sample, with 3 replicates for each sample.
• The plate layout for sample loading can be adjusted based on the sample Quantity

5.2 Seal the 96-well plate with sealing film. Mix it well in microplate shaker, then spin down the reagents for 10 seconds in microcentrifuge and place it on the qPCR instrument.

6, qPCR program setting

NOTE: The following instructions apply only to the ABI7500 instrument with SDS v1.4. If you use a different instrument or software, refer to the applicable instrument or software documentation.

6.1 Create a new document, then in the Assay drop-down list, select Standard Curve (Absolute Quantitation).

6.2 Click New Detector, then enter E.coli-85 in the Name field, select FAM in the Reporter Dye drop-down list and select (none) in the Quencher Dye drop-down list, then click OK.

6.3 Create new detector for E.coli-103, E.coli-220 and E.coli-550, separately as step2.

6.4 Click New Detector, then enter IPC in the Name field. Select VIC in the Reporter Dye drop-down list and select (none) in the Quencher Dye drop-down list, then click OK.

6.5 Select ROX as the passive reference dye, then Click Next.

6.6 Select the applicable set of wells for the samples, then select the corresponding detector for each well.

6.7 Select Finish, and then set thermal-cycling conditions:

6.7.1 Set the thermal cycling reaction volume to 30 μL.

6.7.2 Set the temperature and time as Table 12:

Table 12. qPCR running temperature and time

*Instrument will read the fluorescence signal during this step

6.8 Save the document, then click Start to start the real-time qPCR run.

7, Results analysis

7.1 Select Set up tab,then set tasks for each sample type by clicking on the Task Column drop-down list:

7.1.1 NTC: target DNA detector task = NTC

7.1.2 NCS, test samples = Unknown

7.2 Set up the standard curve as shown in the following table:

Table 13. Settings for Standard curve

7.3 Select the Results tab, then select Amplification Plot.

7.4 In the Data drop-down list, select Delta Rn vs Cycle.

7.5 In the Analysis Settings window, enter the following settings:

7.5.1 Select Manual Ct.

7.5.2 In the Threshold field, enter 0.02 .

7.5.3 Select Automatic Baseline.

7.6 Click the button in the toolbar, then wait the plate analyzing.

7.7 Select the Result tab>>Standard curve tab, then verify the Slope, Intercept and R² values.

7.8 Select the Report tab, then achieve the mean quantity and standard deviation for each sample.

7.9 Select File > > Export > > Results. In the Save as type drop-down list, select Results Export Files, then click Save.

Note: The parameter settings of the result analysis should be based on the specific model and the software version, and generally can also be automatically interpreted by the instrument.

7.10 Set the value of DNA size-85 to be 100%, calculate the percentage of the DNA size of 103, 220 and 550.

7.11 Analyze the Ct value of IPC. Normally, the mean Ct-IPC value of the sample should be within ±1.0 of the NCS Ct-IPC value. If the mean Ct-IPC value of the sample is significantly higher than the Ct-IPC value of the NCS, this indicates that the sample may be inhibitory to the assay. We recommend to test the ERC samples in the same assay, and take the sample recovery rate result as the criterion.

7.12 The Ct value of NTC and NCS should be larger than the mean Ct value of the lowest standard curve concentration or no significant peaks shown in the amplification curve, meanwhile performs normal amplification curve in the VIC signal channel.