Bird Gender(Sex) Identification Kit

Bird  Gender(Sex)  Identification  Kit

Product Composition

Storage and Expiry date

The validity period is 12 months.

Please store the reagent at -20 °C. It is not suitable to freeze and thaw repeatedly. It should be completely thawed at room temperature before use, mixed thoroughly and centrifuged briefly.

Product Description

This kit uses a pair of primers on the CHD gene of the avian sex chromosomes, uses heat-resistant DNA polymerase (Taq enzyme), four monomer nucleotides (dNTPs) and other components, and uses PCR technology to amplify the primers. Bird sex was determined by agarose gel electrophoresis.

This kit is only suitable for gender identification of non-ratites. Most of the existing birds are non-ratites. The only ratites are ostriches, emus, cassowaries, ostriches (American ostriches), and kiwis.

Reagents and items that users need to prepare by themselves

1． DNA extraction kit

2．1.5 ml centrifuge tube, PCR tube

3．Pipettes and tips (to avoid contamination between samples, please use pipette tips with filters)

4．Disposable gloves and protective equipment and tissues

5．Desktop small volume centrifuge (can be equipped with a rotor for centrifuging 1.5 ml centrifuge tubes and PCR tubes)

Precautions

In order to ensure the accuracy of the experimental results and avoid contamination, it is recommended to set up the following four experimental areas from the preparation of the reaction solution to the addition of the test sample and conduct physical isolation. It is strictly prohibited to open reaction tubes containing amplification products in areas 1-3.

Area 1: Preparation and packaging of PCR reaction solutions (the laboratory has positive pressure requirements).

Area 2: Preparation of nucleic acid test samples (the laboratory has positive pressure requirements).

Area 3: Add the test sample to the reaction solution and perform the PCR reaction (the laboratory has positive pressure requirements).

Area 4: Electrophoresis testing ( The laboratory has negative pressure requirements) .

Preparation before use

1．Sample processing ( area 2 )

Use a DNA extraction kit to extract DNA from the test sample. You can refer to the following samples to choose the appropriate kit:

Feather samples: Micro DNA extraction kit (SIMGEN, Cat.No.3102050)

Fresh blood sample: Whole blood DNA mini kit (SIMGEN, Cat.No.3001050)

Dried blood spot samples: Dried blood spot DNA extraction kit (SIMGEN, Cat.No.3012050)

2．Amplification reagent preparation ( area 1 )

Take out the PCR main reaction solution and Taq enzyme from the kit, melt at room temperature, shake and mix, and centrifuge briefly to allow the reagents to settle to the bottom of the tube.

3．Electrophoresis test preparation (area 4)

Configure a 1%~2% agarose gel and conduct electrophoresis detection after PCR amplification is completed.

Steps:

1.       Each test reaction system is prepared as follows:

2.       Prepare reaction system and aliquot (area 1)

Calculate the amount of each reagent component according to the amount of samples to be tested, add it to a 1.5 ml centrifuge tube, mix thoroughly, centrifuge briefly, and add to the set n (n = number of samples + 1 tube of female bird positive control + 1 tube Male bird positive control + 1 tube of negative control) Add 35 μl PCR detection reaction solution to each PCR reaction tube and transfer to the sample processing area (note that due to pipette errors, usually the mixed PCR detection reaction solution is only enough for n samples Divide into n-1 PCR tubes. It is recommended to add one tube when calculating the number of test samples).

* Example: If 4 samples need to be tested, the reagent should be prepared according to the amount of 8 (n+1) PCR detection reaction solutions, that is: bird sex identification PCR main reaction solution 34.6×8=276.8 μl, and then add Taq enzyme 0.4×8=3.2 μl, mix well and divide the PCR detection reaction solution into 7 PCR tubes at 35 μl per tube. Discard the excess PCR detection solution.

3.       Add sample (area 3)

Add 5 μl each of the prepared DNA solution and control substance (usually 1 tube of negative control, 1 tube of female positive control, and 1 tube of male positive control) into the PCR reaction tube, cap the tube tightly, and mark it. Brief centrifugation. Place the reaction tube into the PCR machine and set the PCR reaction conditions according to the following table:

4.      Electrophoretic detection (area 4)

Put the prepared agarose gel into the electrophoresis buffer, add 5 μl of PCR amplification product and 5 μl of control amplification product, electrophores in the electrophoresis buffer for 20 to 25 minutes, then take it out and place it in the gel imager Observe the electrophoresis bands.

Result analysis:

1．Quality control standards

1.1 . The negative control amplification product has no band.

1.2 . The positive control for male birds is one band, and the positive control for female birds is two bands.

If any of the above two points is inconsistent, the experimental results will be invalid.

2．Judgment of results

3.1. If the amplification product has only one bright and clear band, it can be directly judged as a " male bird ".

3.2. If the amplification product has two bright and clear bands, it can be directly judged as a " female bird ".

3.3. If the amplification product has two bands, but one is bright and the other is dim, the test should be repeated or the report should indicate that "the possibility of contamination of the sample submitted for inspection cannot be ruled out."