A method for constructing t vector
A carrier and precursor technology, applied in the field of genetic engineering, can solve the problems of many quality control links, low quality of the T carrier, and cumbersome production process, reducing operation steps and control links, high enzyme digestion yield, and self-cyclization. low rate effect
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Embodiment 1
[0041] Embodiment 1: Amplification of the resistance gene fragment containing AhdI site
[0042] Using the pEGFP-C1 plasmid as a template, use the upstream primer with the nucleotide sequence shown in SEQ ID No: 2 and the downstream primer with the nucleotide sequence shown in SEQ ID No: 3 to carry out PCR amplification to obtain the The kanamycin resistance gene fragment at the AhdI site.
[0043] The PCR amplification reaction system is:
[0044]
[0045] Add sterilized distilled water to make up the total amount of the system to 20 μL.
[0046] The PCR reaction procedure is:
[0047]
[0048] The resulting PCR product was purified and recovered, and 5 μL of 1% agarose gel electrophoresis was used for detection. The results are shown in figure 1
Embodiment 2
[0049] Example 2: Preparation of T carrier precursor
[0050] The PCR product recovered in Example 1 and the pUC-19 plasmid were double-digested with BamHI and HindIII respectively (the endonuclease and corresponding buffer used were Takara products), and the reaction system was as follows:
[0051] Add the following ingredients to Tube A:
[0052]
[0053] Add sterilized distilled water to make up the total amount of the system to 30 μL.
[0054] Add the following ingredients to tube B:
[0055]
[0056] Add sterilized distilled water to make up the total amount of the system to 30 μL.
[0057] Put tubes A and B in a constant temperature water bath at 37°C for 6-8 hours for digestion and digestion, and then perform gel electrophoresis and DNA gel recovery for the digested products. The recovered fragments were ligated overnight at 16°C under the action of T4 DNA ligase (8-12h).
[0058] Connection reaction system:
[0059]
[0060]
[0061] Add sterilized dis...
Embodiment 3
[0063] Embodiment 3: Preparation of T carrier
[0064] The T vector precursor obtained in Example 2 was digested with the specific restriction endonuclease AhdI in the following manner, and the linear vector with a size of about 2000 bp was recovered by electrophoresis, which was the T vector.
[0065] Enzyme digestion reaction system:
[0066]
[0067] Add sterilized distilled water to make up the total amount of the system to 30 μL.
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