Specific molecular marker of wild emmer alpha-amylase inhabiting factor and its utilization
An amylase inhibition and molecular marker technology, which can be used in applications, sugar derivatives, genetic engineering, etc., can solve problems such as differences in inhibitory activity, and achieve the effects of accurate results, fast and efficient detection, and good promotion prospects.
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Embodiment 1
[0052] 1. Genomic DNA extraction:
[0053] Genomic DNA was extracted by CTAB method. Take 2 g of fresh young leaves, grind them into fine powder with liquid nitrogen, add 2×CTAB extract (2% CTAB; 1.4M NaCl, 0.1M Tris-HCl, pH8.0, 0.1MEDTA, pH8.0) preheated to 65°C , add 2% β-mercaptoethanol) 15ml before use, and mix well.
[0054] 65 ℃ water bath for 30-45min, during which gently shake to mix.
[0055] After cooling to room temperature, add an equal volume of chloroform:isoamyl alcohol (24:1), mix gently until the supernatant becomes milky, and centrifuge at 4000rpm for 10min.
[0056] Take the supernatant, add an equal volume of isopropanol, and place in an ice bath to precipitate DNA.
[0057] Hook up the DNA, wash twice with 70% alcohol, wash once with absolute ethanol, air-dry the DNA, and dissolve in an appropriate amount of TE solution with pH 8.0. Add RNase to a final concentration of 100 μg / μl.
[0058] DNA concentration and quality were detected by agarose gel ele...
Embodiment 2
[0103] 1, with the first step of embodiment 1.
[0104] 2. Primer design:
[0105] According to the sequence alignment results of DNAMAN software, the specific primers for specific amplification of the site were used at the 47 bp of the coding region according to the site specificity. The primers are as follows:
[0106] F47: R-terminal primer: 5′ACTCATTT / CGCTTGACTAGGC 3′
[0107] F-terminal primer: 5′AGTACARCGCATGGAGTGG 3′
[0108] F47: length 19bp, GC content 52.6%, molecular weight 5.94kDa, Tm 56.7 degrees
[0109] 3. Gene amplification:
[0110] The PCR reaction system was 5μL of 10×PCR buffer, 1.5U of Ex TaqTM DNA polymerase, 2mmol / LMgCl2, 0.2mmol / L dNTP, 150ng of each primer, 100ng of template DNA, and double distilled water to a total of 50μl. The PCR reaction program was pre-denaturation at 94°C for 4 min; denaturation at 94°C for 45 s, annealing at 57°C for 45 s, extension at 72°C for 1 min, a total of 35 cycles; extension at 72°C for 5 min.
[0111] 4, wit...
Embodiment 3
[0113] 1, with the first step of embodiment 1.
[0114] 2. Primer design:
[0115] According to the sequence alignment results of DNAMAN software, the specific primers for specific amplification of the site were used at the 125 bp coding region according to the site specificity. The primers are as follows:
[0116] F125: R-terminal primer: 5′ACTCATTT / CGCTTGACTAGGC 3′
[0117] F-terminal primer: 5′CTGTCGTCCATTGCTTAG 3′
[0118] F125: length 18bp, GC content 50.0%, molecular weight 5.50kDa, Tm 51.9 degrees
[0119] 3. Gene amplification:
[0120] The PCR reaction system was 5μL of 10×PCR buffer, 1.5U of Ex TaqTM DNA polymerase, 2mmo / LMgCl2, 0.2mmol / L dNTP, 150ng of each primer, 100ng of template DNA, and double distilled water to a total of 50μl. The PCR reaction program was pre-denaturation at 94°C for 4 min; denaturation at 94°C for 45 s, annealing at 62.5°C for 45 s, extension at 72°C for 1 min, a total of 35 cycles; extension at 72°C for 5 min.
[0121] 4, with t...
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