Constructing and applying method of entry vectors pAmpGate and pGentGate
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A carrier and circular carrier technology, applied in the fields of genetic engineering and molecular biology, can solve problems such as unsuitable splicing and cloning, interference, etc., and achieve the effect of saving experimental costs
Active Publication Date: 2016-12-14
INST OF CEREAL & OIL CROPS HEBEI ACAD OF AGRI & FORESTRY SCI
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This scar sequence may interfere with subsequent experiments and is not suitable for splicing clones of some experimental fragments
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Embodiment 1
[0040] Embodiment 1, construction of entry vector pAmpGate and pGentGate
[0041] 1. Construction of pAmpGate vector
[0042] 1. Construction of caR-ccdB-lacZ element
[0043] First design primers Xcm1-Bsa1-Bbs1-F and Xcm1-Bsa1-Bbs1-R, as follows:
[0044] Xcm1-Bsa1-Bbs1-F:
[0045] 5'-TCC CCAatacttgtc c gGAGACCg ca GTCTTC c TCC GAATTCGCTTACTAAAAGCC-3';
[0046] The underlines are the recognition sites of Xcm1, Bsa1, Bbs1 and BamH1 enzymes, two The cohesive ends produced by Bsa1 and Bbs1 enzyme digestion, respectively. Make sure that the 3 consecutive nucleotides in bold are in the reading frame when constructing the fusion protein for the N-terminus.
[0047] Xcm1-Bsa1-Bbs1-R:
[0048] 5'-CC CCAatacttgta c g GAGACC g ca GTCTTC cAGGCCT GA G AAAAAAAAgcggacctcCGAA-3'
[0049] The underlines are the recognition sites of Xcm1, Bsa1, Bbs1 and Xho1 enzymes, two The cohesive ends produced by Bsa1 and Bbs1 enzyme digestion, respectively. Th...
Embodiment 2
[0075] Embodiment 2, the use of entry vector pAmpGate and pGentGate
[0076] Using the pAmpGate or pGentGate entry vector constructed in Example 1, the Golden Gate method can be used to construct the entry clone. Specifically: first, introduce the Bsa1 (or Bbs1) restriction site sequence into the forward primer and reverse primer; then, use high-fidelity DNA polymerase PCR to amplify the target fragment and purify the product; then, use the "two-step method "Recombine the spliced fragment to make it into the entry vector; finally, transform the entry clone into Escherichia coli TOP10 competent and select positive clones for sequencing to complete the construction of the entry clone.
[0077] During the primer design process, the cohesive ends generated by the Bsa1 and Bbs1 cleavage sites designed on the attL1 side and attL2 side of the vector are "ACCT" and "GTAT" respectively (such as figure 1 shown), so the restriction sites of the forward primer and reverse primer at the...
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Abstract
The invention discloses a constructing and applying method of entry vectors pAmpGate and pGentGate. The entry vectors are annular vectors with DNA fragments A, and the 5' ends to the 3' ends of the DNA fragments contain attL1 sequences, identification sequences for IIs-type restriction enzymes A, identification sequences for IIs-type restriction enzymes B, first screening tag sequences, identification sequences for IIs-type restriction enzymes B, identification sequences for IIs-type restriction enzymes A and attL2 sequences. According to the entry vectors, the Golden Gate construction method can be adopted to replace traditional BP reactions to construct entry cloning, expensive BP enzymes can be replaced with the economical IIS-type restriction enzymes and DNA ligase, and the experiment expense is greatly saved; meanwhile, the entry vectors can be used for both single-fragment-DNA recombination cloning and multi-fragment efficient 'seamless' jointing, and the construction and applying method are quite economical, convenient and fast in practical researching.
Description
technical field [0001] The invention belongs to the fields of genetic engineering and molecular biology, and relates to a construction method and application method of a seamless and efficient entry carrier, in particular to a construction method and application of the entry carrier pAmpGate and pGentGate. Background technique [0002] The research of modern molecular biology largely relies on DNA recombination technology, artificial recombination of DNA sequence in vitro is very critical in the whole process of gene cloning. With the development of biotechnology, efficient and economical DNA recombination methods have been paid more and more attention to and pursued by people. [0003] In the DNA recombination technology, the traditional enzyme cutting-ligation classical cloning method is the most conventional method, and it is still widely used. This method uses restriction endonucleases that recognize specific DNA sequences to cut the DNA fragments and carrier sequences ...
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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/65
CPCC12N15/63C12N15/65C12N2800/80
Inventor 张孟臣娜仁杨春燕闫龙史晓蕾邸锐赵鑫冯燕
Owner INST OF CEREAL & OIL CROPS HEBEI ACAD OF AGRI & FORESTRY SCI
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