Method for rapid construction of RNA 3' terminal gene expression library
A gene expression and library technology, applied in the field of molecular biology, can solve the problems that the end of the molecule cannot be introduced by a transposase, the sequence may be the same or different, and the two ends of the 3' region of the mRNA molecule cannot be connected by different adapters, so as to avoid information Lost, the method is simple and easy to implement, and the effect of building a library is high
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[0079] Example 1 Enter the paired-end sequencing adapter by PCR after transposition method
[0080] 1. Enrichment of mRNA
[0081] For the enrichment of eukaryotic mRNA, the specific steps are conventional mRNA enrichment steps. For example, an mRNA purification kit (Beijing Quanshijin Biology, catalog number: EC801) can be used, and the steps are omitted here.
[0082] 2. Synthesis of the first strand cDNA
[0083] For the synthesis of first-strand cDNA, for example, use the first-strand cDNA transcription synthesis kit (Beijing Quanshijin Biology, catalog number: AT301) for this operation:
[0084] 1> Add the components according to the following system:
[0085]
[0086] Oligo(T)-VN1 is an Oligo(dT) reverse transcription primer with a first linker, the sequence is: GACGTGTGCTCTTCCGATCTAAAAAAAAAAAAAAAAAAAAAAAAVN (shown in SEQ ID NO.1), where V represents the three bases of G / A / C Any of the four bases, N represents any of the four bases G / A / C / T.
[0087] 2> Mix gently; incubate at 42°C ...
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[0118] Example 2 Direct sequencing after transposition method
[0119] 1. Enrichment of mRNA
[0120] Same as step 1 in example 1
[0121] 2. Synthesis of the first strand cDNA
[0122] The synthesis of the first strand cDNA is performed. For example, in this example, the first-strand cDNA transcription synthesis kit (Beijing Quanshijin Biology, catalog number: AT301) was used to perform this operation:
[0123] 1> Add the components according to the following system:
[0124]
[0125] Oligo(T)-VN2 is Oligo(dT) reverse transcription primer 2 with a first linker, and its sequence is: CAAGCAGAAGACGGCATACGAGAT[index-i5]GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAAAAAAAAAAAAAAAAAAAAAVN.
[0126] Among them, [index-i5] plays a role in distinguishing the data of each sample, each sample has a different index, and the data of the sample will be distinguished by the difference of the index when sequencing; [index-i5] is inserted in the with Oligo (dT) reverse transcription primer original sequence of th...
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