Paddy rice transgenic detection kit
A detection kit and genetically modified technology, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of time-consuming, labor-intensive and high cost, and achieve the effects of improving sensitivity, simple operation, and firm combination
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Embodiment 1
[0054] The rice transgenic detection kit in this example consists of a primer combination and a membrane chip. The primer combination is a combination of 6 DPO primers (Dual Priming Oligonucleotide, dual priming oligonucleotide primers). The base sequence of each pair of primers is 5'-3' As follows, wherein base I is inosine nucleotide:
[0055] Cry1Ab: forward primer: ATTCAATTCAACGACATGAAIIIIICCTTGAC
[0056] Reverse primer: TTGTAACGGCTATTGATGGTIIIICATCGAA;
[0057] Cry1Ac: forward primer: TTCAGGACCAGGATTCACTGIIIIIIGACCTCG
[0058] Reverse primer: AGGTGAATAGGGGTCACAGAIIIIIIACCTCAC;
[0059] BAR: forward primer: GACGACCTCGTCCGTCTGCGIIIIIGCTATCC
[0060] Reverse primer: CAGCAGGTGGGTGTAGAGCGIIIIICCCAGTC;
[0061] CaMV35S: Forward primer: GATAAAGGAAAGGCCATCGTIIIIIIATGCCTC
[0062] Reverse primer: TGGGATTGTGCGTCATCCCTIIIIICAGTGGA;
[0063] NOS 3': Forward primer: TTAAGATTGAATCCTGTTGCIIIIITTGCGAT
[0064] Reverse primer: CGCGCGCGATAATTTATTCCTIIIIIGCGCGCT;
[0065] gos9: for...
Embodiment 2
[0113] The rice transgenic detection kit in this example consists of a primer set, a membrane chip, excipients and liquid preparation, the liquid preparation is 10×PCR buffer, pre-hybridization liquid, hybridization liquid, washing liquid, blocking liquid, streptavidin-labeled spicy Root peroxidase and tetramethylbenzidine (TMB) chromogenic solution, among which, the prehybridization solution is 5×SSC, 0.1% SDS and 10×Denhardt’s; the hybridization solution is 5×SSC, 0.1% SDS, 5×Denhardt’s , 50% deionized formamide and 100ug / ml yeast tRNA; washing solutions were wash solution 1: 2×SSC and 0.1% SDS, wash solution 2: 0.5×SSC and 0.1% SDS, wash solution 3: 100 mM Tris- HCl, pH 7.5, 150 mM NaCl, Wash 4: 100 mM Tris-HCl, pH 9.5, 100 mM NaCl and 100 mM MgCl 2 ; Blocking solution is 3% BSA, 100 mM Tris-HCl, pH7.5, 150 mM NaCl; Tetramethylbenzidine chromogenic solution is tetramethylbenzidine chromogenic solution A solution: 200mM sodium citrate pH5.4 , 0.2mg / ml tetramethylbenzidine, ...
Embodiment 3
[0118] The rice transgenic detection kit in this example is composed of a primer combination, a membrane chip, excipients and liquid preparation. Among them, one tube of multiple PCR amplification reagent is 600ul, and each 48ul reagent can detect a DNA sample. The amplification system of each 48ul is composed as follows: 10 ×PCR Buffer (Takara) 5ul, dNTP (2.5mM each) 5ul, 6 pairs of multiplex PCR primers (20μM) 0.5ul each, Tag primer (20μM) 3.5ul, EX-Taq Polymerase (Takara, 5U / ul) 0.5ul, wxya 2 O was added to 48ul. For each sample detection, 48ul of the amplification reagent should be aspirated, and 2ul of the genomic DNA to be detected (about 50ng / ul) should be added.
[0119] Positive oligonucleotide single-stranded DNA (10uM) a tube of 15ul.
[0120] A bottle of 30ml prehybridization solution (5×SSC, 0.1% SDS, 10×Denhardt’s). A 30ml bottle of hybridization solution (5×SSC, 0.1% SDS, 5×Denhardt’s, 50% deionized formamide, 100ug / ml yeast tRNA).
[0121] Wash solution 1 (...
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