Reference internal type dual-luciferase reporter vector and application thereof
A dual-luciferase and luciferase technology, applied in the fields of molecular biology and cell biology, can solve the problems of large differences within batches and between batches, increased material costs and labor, and easy to cause misjudgment. , to reduce the experimental cost, improve the repeatability and reliability, and achieve the effect of a wide range of applications
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Embodiment 1
[0081] Example 1 Design and synthesis of chimeric primers for cloning firefly luciferase gene
[0082] The sequence of the firefly luciferase gene comes from the reporter vector pGL3-promoter, and it is planned to be cloned between the Renilla luciferase gene and the ampicillin resistance gene on the reporter vector pRL-TK. The chimeric primer sequences used were:
[0083] pF-Fluc (SEQ ID No: 1)
[0084] 5' AAGGATCCAGGTGGCACTTTTCG
[0085] pR-Fluc (SEQ ID No: 2)
[0086] 5' GAAAAATAAACAAATAGGGGTTCCGCGCAC
[0087]
[0088] P1R (SEQ ID No: 3) 5'CGAAAAGTGCCACCTGGATCCTT3'
[0089] All primers were synthesized by Shanghai Yingjun Biotechnology Co., Ltd., packaged, transported, and stored in the form of dry powder. The primers were diluted with TE buffer (ph=8.0) to a solution of 10 μmol / L, and stored at -20°C for use.
Embodiment 2
[0090] Example 2 One-step binary bridging coupled long-distance PCR amplification of the linear fusion fragment of the firefly luciferase gene and the reporter vector pRL-TK
[0091] i) Sources of reagents and materials
[0092] High-fidelity DNA polymerase KOD plus and its matching buffer (10×buffer), dNTP, magnesium sulfate MgSO 4 All were purchased from Toyobo Biotechnology Co., Ltd., the product number is KOD-201, and stored at -20°C. The reporter vectors pGL3-promoter and pRL-TK were purchased from Promega Company in the United States, extracted and purified according to the instructions of the small-scale ultra-pure plasmid extraction kit provided by Beijing Tiangen Biotechnology Co., Ltd., and stored at -20°C.
[0093] ii) See Table 1 for system composition.
[0094] Table 1 PCR components and concentration
[0095] Element
The initial concentration
Dosage
Final concentration
PCR buffer
10×
5μl
1×
dNTP
2mmol / L
...
Embodiment 3
[0103] Example 3 DpnI digests PCR product
[0104] Restriction endonuclease DpnI specifically recognizes and cuts the methylated GATC sequence, which was purchased from Fermentas Company in Lithuania, Cat. No. ER1702. The composition of the reaction system is shown in Table 2.
[0105] Table 2 Dpn I enzyme digestion system
[0106] Element
The initial concentration
Dosage
Final concentration
Buffer Tango TM
10×
3μl
1×
Dpn I
10units / μl
1μl
0.33unit / μl
PCR product
26μl
total capacity
30μl
[0107] Add the above ingredients one by one on ice and mix thoroughly.
[0108] Incubate in a 37°C water bath for 2 hours, and place on ice for transformation.
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