A method for purifying total mRNA from total RNA using slfn13
一种N端、样品的技术,应用在纯化总mRNA领域,能够解决mRNA质量和完整性难以保证、步骤繁琐、纯化效果不理想等问题
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Embodiment 1
[0034] Expression and purification of embodiment 1SLFN13
[0035] The N-terminal domain of SLFN13 (amino acid sequence 1-355 of human SLFN13, hSLFN13-N, Gene ID: 146857 and amino acid sequence 1-353 of rat SLFN13, rSLFN13-N, Gene ID: 303378; collectively referred to as SLFN13- N) Constructed into pET28 vectors respectively, among which "human SLFN13, Gene ID: 146857", "rat SLFN13, Gene ID: 303378" were derived from the GenBank database established by the National Center for Biotechnology Information (NCBI) in the United States. , transform the plasmid into the Rossetta (DE3) expression strain; pick a single clone and put it into 100ml LB medium with kanamycin and ampicillin double antibody for pre-cultivation. Transfer to 5L of TB medium supplemented with double antibodies and expand the culture at 37°C. When the OD reaches 0.4-0.6, cool down to 17°C and add 80uM IPTG to induce the expression of SLFN13 protein; after low-temperature induction for 16-20h, the bacteria are colle...
Embodiment 2
[0037] The present invention provides a method for purifying total mRNA from total RNA with SLFN13, the specific steps are as follows:
[0038] (1) Total RNA extraction: cells or tissue samples can use the traditional TRIzol-chloroform method to extract complete total RNA (if the sample is special, other applicable methods can be considered, and the quality and integrity of the total RNA should be ensured as much as possible);
[0039] (2) Digestion of tRNA and rRNA. According to the amount of total RNA extracted from the sample and the approximate content ratio of tRNA, rRNA and mRNA, calculate the amount of SLFN13 endonuclease and digestion time that need to be added; the content of tRNA, rRNA and mRNA in the total RNA is 12% respectively , 83% and 3% as examples, if take 10ul total RNA with a concentration of 1ug / ul, add 1ul 50uM SLFN13, add 2ul 10X enzyme digestion buffer, add 7ul ddH 2 O was made into a 20ul enzyme digestion system (that is, 5pmol SLFN13 was used for enz...
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