QPCR kit using the probe method of Drosophila genus (excluding internal reference)

QPCR kit using the probe method of Drosophila genus (excluding internal reference)

Anastrepha Schiner Probe Realtime PCR Kit (without internal control)

QPCR kit using the probe method of Drosophila genus (excluding internal reference)Product and Features:
The detection of Drosophila genus has high specificity, and the cross reactivity rate with other closely related genera or non target species is extremely low, generally less than 1%. By testing multiple species that may be subject to interference, the kit can accurately distinguish between the genus Drosophila and other species, avoiding false positive results.It has the following characteristics:

1. Ready to use, users only need to provide a sample DNA template.

2. Primers and probes have been optimized for high analytical sensitivity, reaching up to 100 copies per reaction.

3. Provide a positive control to distinguish false negative samples.

4. High specificity, primers are designed based on highly conserved regions of Drosophila DNA and will not cross react with other DNA.

5. It can be used for both qualitative and quantitative testing. When used for quantification, its linear range is not less than 5 orders of magnitude.

6. This product is sufficient for 50 fluorescent quantitative PCR reactions using a 20 μ L probe system.

7. This product can only be used for scientific research.

Specifications and Ingredients:

Transportation and storage:Low temperature transportation, stored at -20 ℃, with a shelf life of 12 months.
Bring your own reagent sample DNA.

QPCR kit using the probe method of Drosophila genus (excluding internal reference)Usage:
1、 Dilute standard curve sample(Taking the 6 10 fold dilutions of 10E1-10E6 copies/μ L as an example). Due to the high concentration of the standard substance, the following dilution operations must be carried out in a separate area and must not contaminate the sample or other components of this kit. To increase product stability and avoid the spread of infectious pathogens, this product does not provide live samples as positive controls, only non infectious DNA fragments are provided as positive controls.

1. Mark 6 centrifuge tubes, namely 6, 5, 4, 3, 2, and 1.

2. Add 45 μ L of fluorescent PCR specific template diluent with a core gun tip, preferably using a core gun tip, the same below.

3. Add 5 μ L of 1 × 10E7 copy/μ L positive control (provided by the reagent kit) to tube 6, shake thoroughly for 1 minute, and obtain 1 × 10E6 copy/μ L standard curve sample. Put it on ice for later use.

4. Change the gun head and add 5 μ L of 1 × 10E6 copy/μ L positive control (diluted in the previous step) to tube 5. Shake thoroughly for 1 minute to obtain a standard curve sample of 1 × 10E5 copy/μ L. Put it on ice for later use.

5. Change the gun head and add 5 μ L of 1 × 10E5 copies/μ L positive control (obtained from the previous dilution) to tube 4. Shake thoroughly for 1 minute to obtain a standard curve sample of 1 × 10E4 copies/μ L. Put it on ice for later use.

6. Repeat the above operation until obtaining standard curve samples with 6 dilutions. Put it on ice for later use.

2、 Preparation of Sample DNA

7. If there are N samples, it is best to set N+2 extractions, with the extra one being PC (positive control for sample preparation) and one being NC (negative control for sample preparation). You can use 10 μ L of the fourth dilution obtained in the previous step and add a certain amount of water to make the total volume the same as the required starting volume for each preparation. This can be used as

PC. Additionally, use water as NC.

8. Purify the DNA of the sample using a self selected method. This kit is compatible with most sample DNA extraction kits on the market. You can also choose our company's non extraction nucleic acid release agent.

3、 Probe qPCR reaction (20 μ L system, conducted in the sample preparation room)

9. If quantitative analysis is performed and only one repeat is performed, label N+9 PCR tubes, including N+2 tubes

For the N+2 samples obtained in the previous step, 1 is used as a PCR negative control (using water as a template), and 6 are used for the standard curve. If qualitative analysis is performed and only one repetition is made, label N+4 PCR tubes, of which N+2 are used for the N+2 samples obtained in the previous step, 1 is used for PCR negative control (using water as a template), and 1 is used for PCR positive control (directly using the positive control dilution of tube 4 in step 6 as a template). Below, only quantitative analysis will be used as an example to describe the operational steps.

10. Add each component to the marked tube according to the table below(This table only lists one repetition. The positive control is only set after the sample tube and negative control are set, and the positive control sample should be added after all tubes are covered and stored):

11. Cover the machine and perform PCR according to the following parameters:

4、 Data processing

If this reagent kit is used for quantitative detection, plot a standard curve with the log value of the positive control concentration as the horizontal axis and Ct value as the vertical axis. Calculate the log value of the DNA concentration of the sample from the standard curve based on the Ct value of the sample to be tested, and then calculate its concentration.

13. If this reagent kit is used for qualitative testing and only determines positive or negative, the negative control Ct must not have a numerical value, or the Ct value must be equal to or greater than 40. The positive control must have fluorescence logarithmic growth, typical amplification curve, and Ct value should be less than 40. If the Ct of the test sample is less than 40, it is considered positive. If there is no Ct value, or if it is greater than or equal to 40, it is negative.
Only for scientific research, not for clinical diagnosis! All products are for scientific research purposes only and may not be used for any other purposes such as treatment of humans or animals. We do not provide products or services to any individual. The actual product manual received shall prevail, and the website manual is for reference only.