Linear expression carrier construction method need no clone
A technology of linear expression vector and construction method, which is applied in the field of construction of linear expression vector to achieve the effect of wide application range
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Embodiment 1
[0036] Among them, Taq enzyme (DNA polymerase) was purchased from Huamei Bioengineering Company, plasmid pCMV / myc / cyto / GFP, pEF / myc / nuc were purchased from Invitrogen (USA), T4 DNA ligase (3unit / μl), Vent exo-DNA Polymerase was purchased from New England BioLabs (USA), Pfu DNA polymerase was purchased from Boya Biotechnology Company, T4 Polynucleotide Kinase (T4 Polynucleotide Kinase) was purchased from New England BioLabs (USA), and primers were synthesized by TAGC Company (Sweden) , PCR purification kit was purchased from QIAGENE (Germany). Embodiment 1, construct the linear expression vector containing CMV (cytomegalovirus) promoter fragment A, GFP (green fluorescent protein) fragment B, BGHpA (polyadenylic acid) fragment (comprising terminator) C:
[0037] In this example, the RNA sequence in the primers contained 6 ribonucleotides, and the 5' ends of all primers were phosphorylated. Using the plasmid pCMV / myc / cyto / GFP as a template, the CMV (cytomegalovirus) promoter fra...
Embodiment 2
[0058] A'500ng, B'578.75ng, C'275ng (i.e. molar ratio 1:1:1), use DNA ligation kit (Takara) to connect at room temperature, first add an equal volume of solution II and mix well, then add 2 times the volume The solution I was mixed for 5-10 minutes (solution I and solution II are self-contained solutions in the kit), and the linear expression vector can be obtained, which can be directly used to transfect recipient cells. Example 2: Construction of a linear expression vector containing CMV (cytomegalovirus) promoter fragment A, GFP (green fluorescent protein) fragment B, BGHpA (polyadenylic acid) fragment (comprising terminator) C:
[0059] In this example, the RNA sequence in the primers contains 4 ribonucleotides, the 5' ends of primers 1, 2, 1', 2' are all phosphorylated, and the 5' ends of primers 3 and 4 are not phosphorylated , and the linear expression vector was amplified by PCR for the second time. Its construction process is as follows figure 2 Shown:
[0060] (1...
Embodiment 3
[0079] The above mixture was initially denatured at 94°C for 5 minutes, followed by 30 cycles of 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 1 minute, and finally extended at 72°C for 10 minutes to obtain the amplified linear expression vector D. Embodiment 3: Construct the linear expression vector containing EF-1α promoter fragment A, GFP fragment B, BGHpA fragment (comprising terminator) C:
[0080] Using the plasmid pEF / myc / nuc as a template to amplify the promoter EF-1α sequence, using the plasmid pCMV / myc / cyto / GFP as a template to amplify the GFP fragment and the BGHpA fragment respectively,
[0081] In this example, the RNA sequence in the primers contained 6 ribonucleotides, and the 5' ends of primers 3, 1, 2, 1', 2' and primer 4 were not phosphorylated. After PCR amplification, the three DNA fragments were phosphorylated, and then a linear expression vector D' was obtained through a ligation reaction. Its construction process is as follows image 3 Shown: ...
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