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Standard Operation Procedure (SOP) of 16SrRNA

Standard Operation Procedure (SOP) of 16SrRNA

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Standard Operation Procedure (SOP) of 16SrRNA

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  1. Standard Operation Procedure (SOP) of 16SrRNA By CD Genomics https://www.cd-genomics.com/16S-18S-ITS-Amplicon-Sequencing.html

  2. Detection method: • (a) Extraction of bacterial genomic DNA: • Add 50 μl of sterile water to 200 μl of sterile PCR tube, pick up the purified single colony and then mix thoroughly. Prepare bacteriostatic liquid to 1-2 turbidity, vortex for 15 seconds, and cover well with 100°C boiling water for 10 minutes and place it on ice for 3-5 minutes. Centrifuge at 13,000 rpm for 5 minutes. Take about 30 ul of supernatant for DNA extraction.

  3. Detection method: • (B) PCR amplification of 16SrRNA: 50 μl system • 2×PCR Mix: 25 μl • Upstream Primer: 1μl • Downstream primer: 1μl • Taq DNA Polymerase (5 U/μl): 0.5 μl • DNA template: 1 μl • ddH2O: 21.5μl

  4. 16SrRNA detection primers: • 27F: 5’-AGAGTTTGATC(C/A)TGGCTCAG-3’ • 1492R: 5’-TACGG(C/T)TACCTTGTTACGAC-3’

  5. PCR reaction parameters of 16SrRNA:

  6. (C) Agarose electrophoresis detection: • 1×TAE electrophoresis buffer preparation: • 50 × TAE buffer: Tris base 121g, glacial acetic acid 28.56mL, 0.5mol/LEDTA (pH8.0) 50mL, add deionized water to a constant volume to 500ml for room temperature preservation. Diluted 50 × TAE buffer to 1 × TAE buffer before use.

  7. Preparation of 1% agarose gel: • Weigh 0.3g agarose, add 30mL 1 × TAE running buffer, heat dissolved in a microwave oven, remove and cool with water to 50 °C, add 3μl goldview nucleic acid dye; the sample comb is fixed in the tank, The prepared glue is carefully poured into the glue plate to slowly flatten, and after ensuring that there is no bubble, the glue tank is left at room temperature to completely solidify the glue. • After the glue is completely solidified, the gel is placed in the electrophoresis tank, add 1×TAE electrophoresis buffer to rubber plate and take 4 μl of the product in the gel well and electrophoresed at 100 V for 25 minutes.

  8. Testing: • After above procedures, the electrophoresis gel was observed under UV light and see the band of 16S rRNA gene was located at 1500 bp.

  9. The End • This SOP was filed by CD Genomics for 16SrRNA https://www.cd-genomics.com/16S-18S-ITS-Amplicon-Sequencing.html Thank You!

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