A primer and method for identifying yellow-flowered ox ears and ox ears using EST microsatellite markers
A technology of yellow ox ears and microsatellite markers, which can be used in biochemical equipment and methods, determination/inspection of microorganisms, DNA/RNA fragments, etc., and can solve the problems of few applications and low medicinal value.
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Embodiment 1
[0026] Example 1: Constructing and sequencing the transcriptome library of yellow-flowered ox ears and ox ears
[0027] Firstly, the total RNA of yellow-flowered cow's ear and cow's ear is extracted, and the extracted RNA is subjected to agarose gel electrophoresis, nucleic acid concentration and quality detection, and the total RNA concentration is judged. The mRNA containing poly(A) was enriched and purified by magnetic beads containing Oligo(dT), double-stranded cDNA was reverse transcribed and purified, poly(A) was added using Klenow and sequencing adapters were connected, and agarose gel was used to Gel electrophoresis for the recovery and purification of cDNA fragments of the desired size. The cDNA fragments in the PCR amplification products were used, and the PCR target products were gel-recovered, and the qualified cDNA fragments were purified and detected, and sequenced on Illumina HiseqTM2500 as a cDNA library. The sequencing peak map was read by Base calling softwa...
Embodiment 2
[0032] Embodiment 2: the method for identifying yellow-flowered ox ears and ox ears with the primers determined in embodiment 1
[0033] (1) The extraction of the yellow-flowered cow's ear genome DNA and the cow's ear genome DNA is carried out as follows:
[0034] 1) Preheat the 10mL centrifuge tube with 4mL CTAB extract and 80μl mercaptoethanol in a 65°C water bath;
[0035] 2) Take 1-2g of plant leaves in a mortar, grind an appropriate amount of liquid nitrogen into powder, then transfer to preheated CTAB extract, mix well, keep warm in a water bath at 65°C for 30 minutes, during this period, shake it every 5-6 minutes;
[0036] 3) Cool the warmed sample on ice to room temperature quickly, add an equal volume of chloroform:isoamyl alcohol (24:1), mix thoroughly and centrifuge at 12000rpm for 10min, take 500-1000μL of the supernatant into a 1.5ml centrifuge tube medium (the rest of the supernatant was stored at -20°C for later use);
[0037] 4) Add 100 μL of silica bead sus...
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