PCR detection primer for coix lacryma-jobi Waxy gene mutation, and application thereof
A technology for detecting primers and Coix coix, which is applied in the field of PCR detection primers for Coix coix Waxy gene mutations, can solve the problems of high heterogeneity of Coix coix varieties, unusable, difficult purification and rejuvenation, and variety selection, achieving good stability and accuracy The effect of high sex and accelerated breeding process
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Embodiment 1
[0031] Coix selection:
[0032] (1) Select the mature and full grains of 'Xingren Baike' coix variety, and sow them by direct seeding, with a row spacing of 50 cm and a plant spacing of 30 cm, with a seeding rate of 7 grains per litter to ensure the emergence rate.
[0033] (2) Randomly select 30 Coix seedlings, numbered 1-30, take a small amount of Coix leaves to extract Coix genome DNA, use the extracted Coix genome DNA as a template, and use PCR primers to perform specific PCR amplification reaction;
[0034] The total volume of the PCR reaction system is 25 μL, containing 12.5 μL of Taq PCR MasterMix, 1 μL of 10 μM forward primer, 1 μL of 10 μM reverse primer, ddH 2 O 9.5 μL, DNA template 30-50 μg.
[0035] PCR reaction program: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 50 s, annealing at 65°C for 50 s, extension at 72°C for 5 min, 32 cycles, final extension at 72°C for 10 min, storage at 12°C.
[0036] The PCR products were electrophoresed on 0.8% aga...
Embodiment 2
[0041] Coix selection:
[0042] (1) Select mature and plump seeds of the 'Guangxi Baiyi' variety, and sow them by direct seeding, with a row spacing of 50 cm and a plant spacing of 30 cm, with a seeding rate of 7 grains per litter to ensure the emergence rate, and seedlings should be reserved at the 3-leaf stage.
[0043] (2) Randomly select 10 Coix seedlings, numbered 31-40, take a small amount of Coix leaves to extract Coix genome DNA, use the extracted Coix genome DNA as a template, and use PCR primers to carry out specific PCR amplification reaction;
[0044] The total volume of the PCR reaction system is 25 μL, containing 12.5 μL of Taq PCR MasterMix, 1 μL of 10 μM forward primer, 1 μL of 10 μM reverse primer, ddH 2 O 9.5 μL, DNA template 30-50 μg.
[0045] PCR reaction program: pre-denaturation at 94°C for 3min, denaturation at 94°C for 50s, annealing at 67°C for 50s, extension at 72°C for 5min, 32 cycles, final extension at 72°C for 10min, storage at 12°C.
[0046] Th...
Embodiment 3
[0051] Coix selection:
[0052] (1) Select the mature and plump seeds of the 'An Guo Yi Yi' variety, sow by direct seeding method, row spacing 80cm, plant spacing 50cm, sowing rate 10 grains / litter to ensure emergence rate, 6-leaf stage fixed seedlings and reserved seedlings.
[0053] (2) randomly select 8 Coix seedlings, numbered 40-48, take a small amount of Coix leaves to extract Coix genome DNA, use the extracted Coix genome DNA as a template, and use PCR primers to perform specific PCR amplification reaction;
[0054] The total volume of the PCR reaction system is 25 μL, containing 12.5 μL of Taq PCR Master Mix, 1 μL of 10 μM forward primer, 1 μL of 10 μM reverse primer, ddH 2 O 9.5 μL, DNA template 30-50 μg.
[0055] PCR reaction program: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 60 s, annealing at 69.7°C for 50 s, extension at 72°C for 5 min, 32 cycles, final extension at 72°C for 10 min, storage at 12°C. The PCR products were electrophoresed on 0.8...
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