Anopheles sinensis transgene system
A transgenic, Anopheles mosquito technology, applied in the field of biomedicine, can solve the problems of the environment, mosquito resistance, pollution, etc., and achieve the effect of no environmental pollution
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example 1
[0048] Example 1 Construction of Anopheles sinensis genome library
[0049] 1. Extraction of Anopheles sinensis genomic DNA:
[0050]Take 50 adult mosquitoes of Anopheles sinensis, remove the legs and wings, add the adult mosquitoes into a mortar containing a little liquid nitrogen, and quickly grind the adult mosquitoes into powder. After the powder is completely thawed; add an equal volume of extraction Buffer I (50mM Tris-HCl, pH8.0; 50mM EDTA, pH8.0; 100mM NaCl), divide the above homogenate into sterilized 1.5ml EP tubes, add Equal volume of extraction Buffer II (50mM Tris-HCl, pH8.0; 50mM EDTA, pH8.0; 100mM NaCl; 10% SDS; 50-200μg / mL proteinase K), vortexed and mixed. Water bath at 37°C for 3-4 hours until completely dissolved. Centrifuge at 3000 rpm for 3 minutes at room temperature, and transfer the supernatant to a new EP tube. Add an equal volume of phenol to the above-mentioned EP tubes, shake gently for 1 min, centrifuge at 8000 rpm for 5 min at room temperature,...
example 2
[0052] Cloning of Example Two Anopheles sinensis Vg Promoter
[0053] According to the published Drosophila and Aedes aegypti Vg promoter sequences, design a pair of primers:
[0054] P Vg-6838: 5'-ACC AAC CAA ATG GGA AAT GT-3'
[0055] P Vg-7144: 5'-GAC GAT GAA GAG GAA GAT GA-3'
[0056] Using the Anopheles sinensis genome library as a template, PCR amplification was performed with the designed primers. PCR cycle conditions: 95°C, 5min; 95°C denaturation for 1min, 55°C annealing for 1min, 72°C extension for 90s, 30 cycles; 72°C for 8min . After the PCR product was identified by 1% agarose gel electrophoresis, a fragment with a size of about 400bp was obtained, and the gel was cut and purified with a kit to recover the target fragment (the gel recovery kit was from Shanghai Shenergy Gaming Company); refer to the molecular cloning experiment guide ( The second edition, Sambrook J, et al, translated by Jin Dongyan, 55), with CaCl 2 The intermediate host bacteria DH5α compete...
example 3
[0077] Cloning of Example Three Anopheles sinensis Defensin Coding Sequence
[0078] 1. According to the published Aedes aegypti defensin gene sequence, design a pair of primers:
[0079] Def-1: 5′-GAG AAT TCA TGC AGC CCC TCA CTG TC-3′
[0080] Def-2: 5′-AAG GAT CCT TAA TTC CGG CAG ACG CA-3′
[0081] Use Trizol to extract total RNA of Anopheles sinensis, specific steps:
[0082] Take the total RNA (Note: The EP tubes and tips used in the following steps must be treated with DEPC, and the whole process is carried out in an ultra-clean bench with gloves on, and the reagents at the bottom of the reaction have different degrees of toxicity)
[0083] 1. Take 50 adult Anopheles sinensis mosquitoes, remove the legs and wings, add the adult mosquitoes into a mortar containing a little liquid nitrogen, and quickly grind the adult mosquitoes into powder. Add 1ml of Trizol, transfer to a 1.5ml EP tube, shake to dissolve the precipitate, and let stand at room temperature for 5min.
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