Kit capable of quickly detecting real properties of leather and detecting method of kit
A kit and leather technology, which is applied in the field of kits for rapid detection of the real properties of leather, can solve the problems of sensory identification methods that are difficult to identify leather types, damage consumers' health and interests, and are expensive, and achieve effective supervision of leather products Quality, good economic and social benefits, and high sensitivity
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Embodiment 1
[0040] Example 1 Kit for Rapidly Detecting the True Attributes of Leather
[0041] Contains the following components (for 50 PCR reactions):
[0042] (1) DNA extraction buffer (100mL)
[0043] Weigh 2.00mg CTAB (cetyltrimethylammonium bromide), 1.21mg Tris.HCl (trishydroxymethylaminomethane hydrochloride), 0.58g EDTA 2 Put Na (disodium edetate) and 8.18g NaCl in a volumetric flask, dilute to 100mL with water, and mix well. After sterilizing at 120° C. for 30 minutes, 10.00 mg of protease K (purchased from Dalian Bao Biological Co., Ltd.) and 15.43 g of dithiothreitol (DTT) were added.
[0044] (2) Taq DNA polymerase (25μl)
[0045] Purchase 25 μl of Taq DNA polymerase (125 IU in total) (purchased from Dalian Bao Biology Co., Ltd.).
[0046] (3) PCR reaction solution (1000μl)
[0047] Firstly, primers were prepared, and the prepared primer concentration was 100 nmol / L.
[0048] Primers were prepared according to conventional methods in the art. Synthesis was carried out ...
Embodiment 2
[0064] Example 2 Using the kit of Example 1 to detect genetic components in leather samples
[0065] The detection method comprises the following steps:
[0066] (1) Preparation of sample DNA
[0067] a leather sample
[0068] Cut 50cm 2 The left and right leather samples soaked and sterilized by alcohol were put into a shredder and crushed. Use phosphate buffer solution to soak the crushed leather sample for 12 hours under constant shaking conditions, during which the buffer solution is changed every 2 hours to reduce the content of pigment and salt ions such as chromium ions in the leather sample. After soaking, break the leather sample again, take 3 mL of the broken sample, add 2 mL of DNA extraction buffer, and mix well. Place the centrifuge tube at 65°C for 45-60 minutes, and shake it from time to time to fully lyse the sample. Take it out, add 1mL saturated sodium chloride solution, shake vigorously for 3-5min, place at 0°C for 5-10min, and centrifuge. The supernat...
Embodiment 3
[0098] Embodiment 3 reliability experiment
[0099] For the detection of main varieties of leather samples such as cow leather, sheep leather, and pig leather, this example uses real-time fluorescent PCR detection to verify the reliability of the method in Example 2.
[0100] After the DNA of the leather sample was extracted, a real-time fluorescent PCR detection experiment was carried out on the leather DNA sample. Take 948.0 μl of PCR reaction solution, 6.0 μl of Taq DNA polymerase, and 6.0 μl of probe in a centrifuge tube, mix well, distribute 48.0 μl into eight-tube tubes, and add 2.0 μl containing the target gene to the positive control tube For fragmented DNA samples, add 2.0 μl of control sample DNA without the target gene to the negative control tube, add double distilled water to the blank control tube, and then add 2.0 μl of the leather sample DNA to be analyzed to the remaining reaction tubes (reaction The system is 50 μl), centrifuged and placed on a real-time flu...
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