Tobacco virus-resisting RNAi carrier
A technology of tobacco virus and CMV-RNA1, applied in the field of molecular biology to achieve the effect of preventing viral diseases
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Embodiment 1
[0041] Embodiment 1: Utilize RT-PCR to amplify virus single gene fragment
[0042] 1. Total RNA extraction
[0043]Tobacco plants infected with PVY, CMV, and TMV were collected from the field, and total RNA was extracted from infected tobacco leaves according to the method provided by the TransZol kit (Beijing TransGen Biotech Co., Ltd).
[0044] 2. RT-PCR reaction
[0045] The total plant RNA was used as a template, and PVY-R, CMV-R or TMV-R were used as primers for reverse transcription. The reaction system is as follows:
[0046]
[0047]
[0048] After mixing, incubate at 42°C for 50 minutes, and incubate at 70°C for 15 minutes to stop the reaction.
[0049] Using the obtained reverse transcription product as a template, single-fragment PCR amplification was performed.
[0050] The PVY fragment PCR amplification system is as follows:
[0051]
[0052] The CMV fragment PCR amplification system is as follows:
[0053]
[0054] TMV fragment PCR amplification...
Embodiment 2
[0059] Embodiment 2: Construction of the reverse fragment
[0060] 1. Perform 1% agarose gel electrophoresis on all PVY amplification products. Gel was cut and recovered using EasyPure Quick Gel Extraction Kit (TransGen Biotech, China). The fragment recovery product was connected to the pMD18-T simple vector, overnight at 4°C, and the connection system was as follows:
[0061]
[0062] 2. The ligation product was transformed into DH5α competent cells by heat shock method;
[0063] 3. Pick a single colony and shake it in liquid LB medium containing ampicillin at 37°C;
[0064] 4. Plasmid extraction by alkaline lysis;
[0065] 5. Use primers PVY-F and PVY-R to screen positive clones by PCR;
[0066] 6. Carry out double enzyme digestion on the identified positive recombinant plasmid. The enzyme digestion system is as follows:
[0067]
[0068] Electrophoresis, cutting the gel to recover the band around 3300bp;
[0069] 7. Take 5.0 μL of the CMV amplification product a...
Embodiment 3
[0086] Example 3: Amplification of forward fragments
[0087] 1. Using pMD18S-RPCT as a template, PCR amplifies the forward fragment F-PCT,
[0088] The system is as follows:
[0089]
[0090] The procedure is as follows:
[0091]
[0092] 2. Electrophoresis, gel cutting and recovery of 800bp fragments (F-PCT);
[0093] 3. Connect the recovered product of F-PCT to the pGEM-T easy (Promega, America) vector at 4°C overnight. The connection system is as follows:
[0094]
[0095] 2. The ligation product was transformed into DH5α competent cells by heat shock method;
[0096] 3. Pick a single colony and shake it in liquid LB medium containing ampicillin at 37°C;
[0097] 4. Plasmid extraction by alkaline lysis;
[0098] 5. PCR screens out the clones connected to F-PCT (system and program are the same as the amplification process of F-PCT fragments);
[0099] 6. BamHI / SpeI double enzyme digestion to identify the direction of the inserted clone. The enzyme digestion ...
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