Microbial genome DNA (deoxyribonucleic acid) indirect extraction method for evaluating diversity of animal intestinal microflora
A technology of intestinal microorganisms and extraction methods, which is applied in the field of indirect extraction of microbial genome DNA, can solve the problems of low DNA recovery rate and difficult removal of pollutants, and achieve the effects of low cost, strong applicability, and simple operation
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Embodiment 1
[0036] 1) Add 0.5mL of pre-cooled sterile water to 0.5g of test mouse feces samples, mix well, vortex for 10 minutes, then add 0.5mL homogenization buffer A, vortex for 10 minutes, low speed (250g) Centrifuge at room temperature for 10 minutes, collect the supernatant and transfer to another centrifuge bottle.
[0037] 2) Add 1 mL of pre-cooled homogenization buffer B to the precipitate obtained in the previous step to wash and resuspend, vortex for 10 minutes, centrifuge at low speed (250g) for 10 minutes at room temperature, take the supernatant, and use the supernatant obtained in step 1) Combine and centrifuge at 10,000 rpm for 30 minutes at room temperature to recover bacterial cells, and discard the supernatant.
[0038] 3) Add 1.5ml of washing solution C to the bacterial cells obtained in the previous step, wash for 5 minutes, and centrifuge at 10,000 rpm for 30 minutes at room temperature to pellet the bacterial cells. Add 160ul lysozyme (50mg / ml) and 20ul proteinase K ...
Embodiment 2
[0049] 1) Add 0.5mL pre-cooled sterile water to 0.5g test rat feces sample, mix well, vortex for 10 minutes, then add 0.5mL homogenization buffer A, vortex for 10 minutes, low speed (250g) at room temperature Centrifuge for 10 minutes, collect the supernatant and transfer to another centrifuge bottle.
[0050] 2) Add 1 mL of pre-cooled homogenization buffer B to the precipitate obtained in the previous step to wash and resuspend, vortex for 10 minutes, centrifuge at low speed (250g) for 10 minutes at room temperature, take the supernatant, and use the supernatant obtained in step 1) Combine and centrifuge at 10,000 rpm for 30 minutes at room temperature to recover bacterial cells, and discard the supernatant.
[0051] 3) Add 1.5ml of washing solution C to the bacterial cells obtained in the previous step, wash for 5 minutes, and centrifuge at 10,000 rpm for 30 minutes at room temperature to pellet the bacterial cells. Add 160ul lysozyme (50mg / ml) and 20ul proteinase K (20mg / m...
Embodiment 3
[0062] 1) Add 0.5mL pre-cooled sterile water to 0.5g test white rabbit feces sample, mix well, vortex for 10 minutes, then add 0.5mL homogenization buffer A, vortex for 10 minutes, low speed (250g) at room temperature Centrifuge for 10 minutes, collect the supernatant and transfer to another centrifuge bottle.
[0063] 2) Add 1 mL of pre-cooled homogenization buffer B to the precipitate obtained in the previous step to wash and resuspend, vortex for 10 minutes, centrifuge at low speed (250g) for 10 minutes at room temperature, take the supernatant, and use the supernatant obtained in step 1) Combine and centrifuge at 10,000 rpm for 30 minutes at room temperature to recover bacterial cells, and discard the supernatant.
[0064] 3) Add 1.5ml of washing solution C to the bacterial cells obtained in the previous step, wash for 5 minutes, and centrifuge at 10,000 rpm for 30 minutes at room temperature to pellet the bacterial cells. Add 160ul lysozyme (50mg / ml) and 20ul proteinase ...
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