Construction and application of plant transient expression vector
A transient expression and plant technology, applied in vectors, nucleic acid vectors, and the use of vectors to introduce foreign genetic material, etc.
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Embodiment 1
[0029] The construction of embodiment 1 pEAQE carrier
[0030] (1), using the plant expression vector pEAQ as the basic skeleton, performing 2-hour digestion with two restriction sites of PacI and XhoI, and then recovering a 9200bp fragment by DNA gel electrophoresis;
[0031] (2), synthesis contains artificial sequence as shown in SEQ ID No.1, including 5'UTR region, MCS region, SEKDEL sequence;
[0032] (3), design amplification primers for amplifying the artificial sequence gene fragment, the upstream primer is as shown in SEQ ID No.2, and the downstream primer is as shown in SEQ ID No.3;
[0033] (4) KOD One of TOYOBO TM PCRMasterMix for amplification reaction, the amplification system is: 22ul ddH 2 O. 25ul KOD One TM PCRMasterMix, 1ul 10uM upstream primer, 1ul 10uM downstream primer, 1ul 100ng / ul plasmid template containing the artificially synthesized sequence SEQ ID No.1. PCR amplification program: 98 degrees for 30 seconds; 28 cycles: 98 degrees for 10 seconds; ...
Embodiment 2
[0042] Embodiment 2 inserts GFP gene on the pEAQE vector
[0043] (1), using the plant expression vector pEAQE as the basic skeleton, using the SmaI restriction site for 2 hours of digestion, and then recovering a 10469bp fragment by DNA gel electrophoresis;
[0044] (2), design amplification primers for amplifying artificial sequence gene fragments, upstream primer SEQ ID No.4, downstream primer SEQ ID No.5;
[0045] (3) KOD One of TOYOBO TM PCRMasterMix for amplification reaction, the amplification system is: 22ul ddH 2 O. 25ul KOD One TM PCRMasterMix, 1ul 10uM upstream primer, 1ul 10uM downstream primer, 1ul 100ng / ul plasmid template containing GFP. PCR amplification program: 98 degrees for 30 seconds; 28 cycles: 98 degrees for 10 seconds; 55 degrees for 30 seconds; 68 degrees for 10 seconds; the last 68 degrees for 5 minutes; 4 degrees for 10 minutes;
[0046] (4) The final amplified gene fragments are recovered by gel electrophoresis through gel electrophoresis; sin...
Embodiment 3
[0054] Example 3 Infiltration
[0055] (1) The pEAQE-GFP plasmid was introduced into the competent Agrobacterium by electric shock method, and spread on the Kana and rifampicin double-antibody medium.
[0056] (2), pick the single clone and shake the bacteria with the culture medium, wait for the OD 600 When =0.8, with 8000rpm centrifugation 2 minutes, the bacterium liquid of precipitation is washed with infiltration buffer (10mM MES, 10mM Mgcl 2 , 1uM acetosyringone) resuspended and adjusted OD 600 = 0.6.
[0057] (3) Infiltrate the bacterial solution into the tobacco leaves with a negative pressure vacuum pump, and observe the fluorescence after 2 days.
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