Identification method of regulatory element for regulating transcriptional activity of FATP1 gene promoter
A technology of regulatory elements and promoters, applied in biochemical equipment and methods, recombinant DNA technology, measurement/inspection of microorganisms, etc., can solve problems such as the transcriptional regulation mechanism of bovine FATP1 gene that has not been reported
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[0028] 1. Analysis of luciferase activity of FATP1 gene promoter
[0029] 1. Design of promoter primers
[0030] According to the 5′UTR sequence of the FATP1 gene in GeneBank, the transcription start site identified by 5′-RACE was +1, and gene-specific primers were designed by Clone Manage software:
[0031] The upstream primer is Forward Primer F1:5′-CTACTGTGGTGGGCACTTG-3′
[0032] The downstream primer is Reverse Primer R1: 5'-TTGTTCCCTGGCTGACCTGGAG-3'.
[0033] Bovine genomic DNA was used as a template for PCR amplification, and the target fragment was 2046bp in total from the promoter sequence -1856 to +190. Add 1.2 μL (100ng) DNA template, 0.4 μL KOD-Plus-Ver 2.0 (1U / μL), 2 μL 10×PCR Buffer, 2 μL dNTP (2 mmol / L), 0.8 μL MgSO in 200 μL PCR tube 4 (25mol / L), 0.6 μL ForwardPrimer F1 (10 μM), 0.6 μL Reverse Primer R1 (10 μM), 12.4 μL ddH 2 O, a total of 20 μL, vortexed and centrifuged, placed in a PCR instrument for reaction, the reaction conditions are as follows: pre-de...
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