Construction method for double enzyme digestion simplified genome next generation sequencing library and matched kit
A technology for a second-generation sequencing library and a construction method, which is applied in the field of supporting kits for the construction of second-generation sequencing libraries, can solve the problems of cumbersome process, restricted library construction cost, and high time consumption, and achieves simplified library construction process, reduced synthesis cost, The effect of increasing flexibility
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Embodiment 1
[0136] Double enzyme digestion simplifies the basic operation process of genome next-generation sequencing library construction:
[0137] The first step, the extraction of genomic DNA: using the improved CTAB method to extract the genomic DNA of fresh young bamboo leaves,
[0138] The second step, enzyme digestion of genomic DNA: use two common restriction endonucleases to digest genomic DNA to obtain DNA fragments with corresponding restriction enzyme sites at both ends;
[0139] The genomic DNA has an obvious main band detected by agarose gel electrophoresis and the 260nm / 280nm value of the trace spectrophotometric purity test is between 1.8-2.2, and is diluted to 40-60ng / μl for use;
[0140] The common restriction endonuclease is preferably a restriction endonuclease that recognizes a 4.5 base sequence and a restriction endonuclease that recognizes a 4 base sequence, more preferably AvaII and MspI;
[0141] The third step is to use T4 DNA ligase to connect the A1 adapter (...
Embodiment 2
[0188] The establishment of double enzyme digestion simplified genome next-generation sequencing library construction method:
[0189] This embodiment selects two kinds of bamboos of Gramineae Bambooideae, Phyllostachysviridi-glaucescens (Carr.) A.etC.Riv. and Pseudosasajaponica (Sieb.etZucc.) Makino) as Experimental materials, double enzyme digestion simplified genome next-generation sequencing library construction basic operation process, see figure 1 , including the following steps:
[0190] The first step, genomic DNA extraction
[0191] The whole genome DNA of the young leaves of P. viridi-glaucescens (Carr.) A. et C. Riv. and P. japonica (Sieb. et Zucc.) Makino) were extracted by the improved CTAB method, respectively. Proceed as follows:
[0192] (1) Take 10ml of 4% CTAB and preheat it at 60°C, add 2‰ dithiothreitol DTT and mix well.
[0193] (2) Weigh 60mg of fresh young leaves, add liquid nitrogen and quickly grind them into powder, transfer to a 2ml small centri...
Embodiment 3
[0263] Genome-wide interspecific variation SNP detection of 20 temperate woody bamboo species:
[0264] Use double-digestion simplified genome next-generation sequencing library construction kit for research. Double Digestion Simplified Genome Next Generation Sequencing Library Construction Kit contains:
[0265] 1) A1 linker sequence pairs consisting of 20 sequences, each sequence structure is:
[0266] Positive strand: 5'TACACGACGCTCTTCCGATCTXXXXX3',
[0267] Negative strand: 5'GWCYYYYYAGATCGGAAGAGCGTCGTGTA3'.
[0268] Wherein: XXXXX in the positive strand represents the DNA barcode sequence, YYYYY in the negative strand represents the sequence complementary to the DNA barcode, and W represents the base A or T. This kit only provides the A1 linker sequence pair corresponding to AvaII, and the A1 linker sequence pair corresponding to the rest of the enzymes can be directly synthesized from biological companies.
[0269] 2) A2 linker sequence, specifically:
[0270] Posit...
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