Specific primer, kit and identification method for identifying donkey-derived components
A donkey-derived, kit-based technology, applied in the field of identification of Chinese herbal medicines, can solve the problems of less DNA content, DNA damage, and short fragments, and achieve the effects of accurate identification results, high specificity, and small molecular weight
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Embodiment 2
[0173] Example 2 Kit
[0174] This embodiment provides a kit for identifying donkey-derived components, each of which includes a primer solution part and a PCR reaction solution part that are individually packaged; The specific primers; the PCR reaction solution part contains amplification buffer, dNTPs, Taq DNA polymerase and sterile ultrapure water for PCR amplification, and the formula of each kit is:
[0175] DNA template, 50ng / μL, 3μL;
[0176] Upstream primer, 10 μM, 1 μL;
[0177] Downstream primer, 10 μM, 1 μL;
[0178] Taq DNA polymerase, 5U / μL, 0.4μL;
[0179] dNTPs, 10mM, 0.5μL;
[0180] 10×PCR amplification buffer, 2.5 μL;
[0181] Make up to 25 μL with sterile ultrapure water.
Embodiment 3
[0182] Embodiment 3 identification method
[0183] This example provides a method for identifying donkey-derived components using the specific primers of Example 1 or the kit of Example 2, including the following steps:
[0184] (1) Ejiao to be tested is pretreated with Ni-NTA
[0185] Take 50mg donkey-hide gelatin sample 1, add 5ml Lysis Buffer (NaH 2 PO 4 50mM, NaCl 300mM, NaOH to adjust the pH to 8.0), warm the bath until the sample melts completely, put it in a Ni-NTA prepacked column (the amount of filler is based on 10mg protein per 1ml Ni-NTA filler), shake on ice Combine for 1 hour, collect the breakthrough solution, and evaporate to dryness at 85°C to obtain the pretreated sample.
[0186] (2) DNA extraction
[0187] Take the pretreated sample and use the TAKARA MiniBest DNA kit to extract the total DNA, operate according to the instructions, and the extraction steps are the same as the aforementioned TAKARA MiniBest DNA kit.
[0188] (3) PCR amplification
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Embodiment 4
[0203] Embodiment 4 identification method
[0204] This example provides a method for identifying donkey-derived components using the specific primers of Example 1 or the kit of Example 2, including the following steps:
[0205] (1) Ejiao to be tested is pretreated with Ni-NTA
[0206] Take 100mg donkey-hide gelatin sample 2, add 1ml Lysis Buffer (NaH 2 PO 4 75mM, NaCl 100mM, NaOH to adjust the pH to 10.0), warm the bath until the sample melts completely, place it in a Ni-NTA prepacked column (the amount of filler is based on 8mg protein per 1ml Ni-NTA filler), shake on ice Combine for 2 hours, collect the breakthrough solution, and evaporate to dryness at 80°C to obtain the pretreated sample.
[0207] (2) DNA extraction
[0208] Take the pretreated sample and use the TAKARA MiniBest DNA kit to extract the total DNA, operate according to the instructions, and the extraction steps are the same as the aforementioned TAKARA MiniBest DNA kit.
[0209] (3) PCR amplification ...
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