Geranyl pyrophosphate synthase gene from anoectochilus roxburghii and application of geranyl pyrophosphate synthase gene from anoectochilus roxburghii
A technology of geranyl pyrophosphate and geranyl pyrophosphate, applied in the field of genetic engineering, to achieve the effect of easy amplification and simple operation
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Embodiment 1
[0026] This embodiment provides the extraction of RNA from the total leaves of Anoectochilus, using the Trizol extraction kit of Dalian Bao Biocable Company, which includes the following steps:
[0027] (1) 100 mg of fresh leaves of Anoectochilus are grinded into powder and put into (1) 1.5 mL centrifuge tube, then immediately add 1 mL of RNAiso Plus, and mix by inversion to obtain a homogenate.
[0028] (2) The well-mixed homogenate was allowed to stand at room temperature for 5 minutes, and then centrifuged at 12000g at 4°C for 5 minutes.
[0029] (3) Transfer 800ml of the supernatant to a new centrifuge tube. Do not aspirate the precipitate. Then add 200ml of chloroform to the supernatant, shake vigorously until the homogenate emulsifies and become milky white, and then let it stand at room temperature for 5 minutes to obtain a mixture liquid.
[0030] (4) Centrifuge the mixed solution at 12000g at 4°C for 15 minutes. At this time, the homogenate is divided into three layers. From...
Embodiment 2
[0036] This embodiment provides a transcriptome sequencing method of Anoectochilus Fujian, which includes the following steps:
[0037] Extract the total RNA from the leaves of Anoectochilus fujianensis, check the quality of RNA extraction, and meet the requirements of building a database (RNA concentration> 250ng / μL, total amount> 20μg, OD260 / OD280 between 1.8~2.2, good integrity, RIN> 6.5). Then, use magnetic beads to enrich poly(A)mRNA to break into fragments. Using this as a template, the first cDNA strand and the second cDNA strand are sequentially synthesized, after purification, elution, end repair, and poly(A) ) Then connect the sequencing adapter. Select 200bp~700bp size fragments for PCR amplification, establish cDNA sequencing library, and use IIIumina HiSeq 2000 for sequencing. This part of the experiment was commissioned by Main Technology Services Cable.
[0038] Use the short reads assembly software Trinity (v2.4.0) for De novo assembly, and after obtaining the Con...
Embodiment 3
[0040] This embodiment provides a method for synthesizing the first strand of cDNA of Anoectochilus fujianensis, which includes the following steps:
[0041] The purified total RNA of Anoectochilus leaves obtained in Example 1 was used as a template, oligo(dT)18 was used as a reverse transcription primer, and PrimeScriptTM Reverse Transcriptase (TakaRa China) was used according to SMART. TM PCR cDNA Synthesis Kit (Clontech USA) instructions for the synthesis of the first strand of cDNA. The total volume of the reaction system is 20 μL.
[0042] (1) Prepare reverse transcription mixture 1 in a 0.2mL PE tube according to Table 1 below;
[0043] (2) Prepare reverse transcription mixture 2 in another 0.2mL PE tube according to the reagents in Table 1 below;
[0044] (3) The reverse transcription mixture 1 in step (1) is incubated at 65°C for 5 minutes and then quickly chilled on ice for 2 minutes, and centrifuged for a few seconds to make the mixture of template RNA, primers, etc., gathe...
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