Primer for identifying authenticity of donkey-hide gelatin, reagent kit and detection method of primer
A detection kit, donkey-hide gelatin technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve problems such as failure to identify, achieve convenient use, good specificity and high accuracy Effect
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Embodiment 1
[0029] 1. Materials and Reagents
[0030] Reagent: Taq Premix Enzyme Premix Ex-Taq TM Hot Start Version kit, DNA markers as molecular weight standard 20bp DNA Ladder Marker were purchased from Treasure Bioengineering (Dalian) Co., Ltd.; DNA fluorescent dye GelRedTM Nuleic Acid Gel Stain 10000×in water was purchased from Biotium Company (Cat. No. 41003) ; Acrylamide and N, N-methylenebisacrylamide were purchased from Beijing Dingguo Biotechnology Co., Ltd.; 5× TBE buffer and 6× Glycerol DNA Loading Buffer were purchased from Sangon Bioengineering (Shanghai) Co., Ltd. Company; primer sequences were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.
[0031] Materials: Horse hide glue, donkey hide glue and mule hide glue are obtained by boiling horse hide, donkey hide and mule hide respectively.
[0032] 2. Detection method
[0033] Extract DNA from horse hide glue, donkey hide glue and mule hide glue respectively to obtain a DNA extraction solution for detection, dilu...
Embodiment 2
[0036] Using the extracted DNA from horse hide glue, donkey hide glue and mule hide glue as templates (final concentration: 10ng / μL), the nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2 were used as primers for PCR amplification. increase. Both upstream and downstream primers were diluted to 100 μM with sterile water, aliquoted into 5 μL / tube, stored at -20 ℃, and diluted to 10 μM before use. Before adding the sample, make a mixture of upstream and downstream primers, specifically 4 μL of 10 μM upstream primer and 4 μL of 10 μM downstream primer, add 52 μL of sterile water, and mix well. The PCR reaction system is 10 μL, and the specific loading conditions are as follows: Premix Ex-Taq TM Hot Start Version: 2.5 μL, Primer Mixture: 1.5 μL, Extracted DNA Diluent 1 μL. PCR amplification conditions were: pre-denaturation at 94°C for 6 min; denaturation at 94°C for 30 s, annealing at 45°C for 30 s, extension at 72°C for 30 s, 35 cycles; extension at 72°C for 7 min; and...
Embodiment 3
[0041] DNA extracted from horse hide glue was used as a template, and the final concentrations were 10 ng / μL, 5 ng / μL, 2.5 ng / μL, 1 ng / μL, and 10 ng / μL, respectively. -1 ng / μL, 10 -2 ng / μL, 10 -3 ng / μL, 10 -4 ng / μL and 10 -5 ng / μL, set the PCR annealing temperature to 45°C, and perform PCR amplification with the same conditions as in Example 2. After the PCR reaction, use 8% polyacrylamide gel (PAGE) for electrophoresis detection, take 2.5 μL PCR product and 0.5 μL 6× Glycerol DNA Loading Buffer and mix evenly, after loading, electrophoresis at 100 V for 40 min, take out the gel , put in 10 mL Gel Red solution (45 mL distilled water, add 5 mL 1mol / L NaCl solution, add 10 μL Gel Red TM Nuleic Acid Gel Stain 10000×in water (mixed evenly) on a shaker for 30 min, rinse the gel with distilled water, and observe it under a gel imager. The results are as follows: image 3 shown. Depend on image 3 It can be seen that when the concentration of DNA template is greater than...
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