Kit for detecting and evaluating genes of children obesity
A gene detection and kit technology is applied in the field of kits for the detection and evaluation of children's obesity physique to achieve the effect of facilitating popularization and application.
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Embodiment 1
[0059] Kit preparation. Including (1) probe preparation; (2) DNA extraction reagent; (3) premixed reaction solution; (4) standard positive control; (5) standard negative control.
[0060] (1) Preparation of the probe;
[0061] Synthesized by a common nucleotide primer synthesizer. The sequence is as follows:
[0062] The sequence of the amplification primers for detecting the PPARG gene is as follows:
[0063] Upstream primer F1(A)C 5-TCT GGG AGA TTC TCC TAT TGA CC-3 23bp,
[0064] Upstream primer F1(A)G 5-TCT GGG AGA TTC TCC TAT TGA CG-3 23bp,
[0065] Downstream primer R1 3-TCA AGG AAG GTC TAT GCC GAT AAC-5 24bp;
[0066] The sequence of the amplification primers for detecting the PPARA gene is as follows:
[0067] Upstream primer F2C 5-CAG TAT TGT CGA TTT CAC AAG TTC C-3 25bp,
[0068] Upstream primer F2G 5-CAG TAT TGT CGA TTT CAC AAG TTC G-3 25bp,
[0069] Downstream primer R2 5-GGA ACT GAG GAA CGA ACT GGG-3 21bp;
[0070] Before use, dilute each OD primer to a co...
Embodiment 2
[0111] Embodiment 2: the use of kit
[0112] (1) Sample preparation
[0113] Sampling method: Use a cotton swab to wipe the inside of the cheek 10 times.
[0114] Note: In order to ensure that the sample is not contaminated by food or drink, do not eat or drink within 30 minutes before sampling.
[0115] a) Processing materials: transfer the cotton swab wiped inside the cheeks into a 2ml centrifuge tube, cut the cotton swab part from its stem with scissors, and add 400 μl buffer GA.
[0116] b) Add 20 μl of proteinase K solution, vortex for 10 seconds to mix, and place at 56° C. for 60 minutes, during which time, vortex and mix several times every 15 minutes.
[0117] c) Add 400 μl buffer GB, mix thoroughly by inversion, and place at 70°C for 10 minutes. At this time, the solution should become clear, and briefly centrifuge to remove the liquid droplets on the inner wall of the tube cap.
[0118] d) Add 200 μl of absolute ethanol, mix thoroughly by inversion, and briefly ce...
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