Scylla paramamosain double-stranded specific deoxyribonuclease as well as preparation method and application thereof
A technology for deoxyribonucleic acid and Scylla simulans, which is applied in the field of Scylla simulatum double-strand specific deoxyribonuclease and its preparation, and can solve the problems of low enzyme cleavage activity and the like
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Embodiment 1
[0107] The extraction of the total RNA of embodiment 1 mud crab
[0108] Extraction of total RNA and synthesis of cDNA: Isolate the hepatopancreas of Scylla pseudocavena, extract the total RNA of the hepatopancreas by RDP method, extract with phenol / chloroform / isoamyl alcohol to remove impurities such as proteins, and obtain the total RNA of the hepatopancreas with good quality (see figure 1 ), the bands of 18s rRNA and 28s rRNA were clear; the OD of total RNA was measured by UV spectrophotometer 260 / OD 280 =2.0, the total RNA can be used for follow-up experiments.
Embodiment 2c
[0109] Synthesis of the first strand of embodiment 2cDNA
[0110] Take 3 μg of RNA and treat it with DNase I first, then add 1 μL of 3’CDS primer to the 3’RACE template; add 1 μL of 5’CDS primer (10 μmol / L) and 1 μL of SMART II Oligo (10 μmol / L) to the 5’RACE template ) for reverse transcription to synthesize the first strand of cDNA.
Embodiment 3
[0111] Embodiment 3SMART II RACE technology obtains result
[0112] The first strand of 3'RACE cDNA and the first strand of 5'RACE cDNA were used as templates, and 3'RACE outer and UPM, 5'RACE outer and UPM were used as primers at both ends to carry out the first round of SMART II RACE amplification. Then use the 50-fold dilution of the first round of RACE-PCR product as a template, and use 3'RACE inner and UPM, 5'RACE inner and UPM as primers at both ends to carry out the second round of SMART II RACE amplification to obtain 3'RACE And 5'RACE results, found that 3'RACE has a specific band at 907bp, and 5'RACE has a specific band at 698bp (see figure 2 and 3 ), recover the two bands, clone and sequence the recovered target bands.
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