Primer for detecting CAMV35S genes, relevant kit and detecting method
A gene detection and kit technology, which is applied in the field of detection and detection, achieves the effects of high yield, low detection cost and high sensitivity
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Embodiment 1
[0040] Example 1 Preparation of Transgenic Crop CAMV35S Gene Rapid Diagnostic Kit
[0041] (1) Synthesize oligodeoxynucleic acid primers by DNA synthesizer according to the following sequence:
[0042] Outer primer F3, the nucleotide sequence of which is shown in SEQ ID NO: 1 or 5;
[0043]Outer primer B3, the nucleotide sequence of which is shown in SEQ ID NO: 2 or 6;
[0044] Internal primer FIP, its nucleotide sequence is shown in SEQ ID NO:3 or 7;
[0045] Internal primer BIP, its nucleotide sequence is shown in SEQ ID NO:4 or 8.
[0046] (2) Purchasing DNA polymerase: Bst DNA polymerase (large fragment) in container.
[0047] (3) Prepare the reaction solution: The formula of the reaction solution contains 2mmol dNTP, 25mmol Tris-Cl, 12.5mmol potassium chloride, 12.5mmol ammonium sulfate, 10mmol magnesium sulfate, 1.25ml TritonX-100, 1mol betaine, 2 mol each of primers FIP / BIP and 0.25 mol each of outer primers F3 / B3 were prepared and placed in containers.
[004...
Embodiment 2
[0058] Example 2 Application of Transgenic Crop CAMV35S Gene Rapid Diagnostic Kit
[0059] In this example, the CAMV35S gene rapid diagnosis kit for transgenic crops prepared in Example 1 is used for the rapid diagnosis of the CAMV35S gene in the sample to be tested.
[0060] 1. Sample processing (template DNA extraction)
[0061] 1) Grind 100mg of the pretreated sample fully into powder in liquid nitrogen and add 700μL CTAB extraction buffer (the sample that does not need to be ground is directly added), shaken and mixed, and kept at 65°C for 30 minutes, during which time, gently invert and mix 2-3 times.
[0062] 2) Add 700 μ L of chloroform-isoamyl alcohol, and gently invert the solution 2-3 times. Centrifuge at 12000g for 5 min to separate the phases.
[0063] 3) Transfer the supernatant to a clean centrifuge tube, add 0.6 times the volume of 4°C pre-cooled isopropanol, let stand at -20°C for 5 minutes, and centrifuge at 12 000 g for 5 minutes.
[0064] 4) Discard ...
Embodiment 3
[0074] Example 3 Specificity Verification of Transgenic Crop CAMV35S Gene Rapid Diagnostic Kit
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