The V6–V8 region of the 16S rRNA gene was amplified using universal (degenerate) PCR primers 799F (acCMGGATTAGATACCCKG) and bac1193R (CRTCCMCACCTTCCTC). Amplification was performed using the HotStarTaq Plus Master Mix Kit (Qiagen, Germantown, MD, USA) under the following conditions: 94 °C for 3 min, followed by 28 cycles of 94 °C for 30 s, 53 °C for 40 s and 72 °C for 1 min, with a final elongation step at 72 °C for 5 min. PCR products were confirmed using a 2% agarose gel. PCR products were then used to prepare DNA libraries following Illumina MiSeq DNA library preparation protocol. Sequencing was performed at the University of Arizona Genetics Core (UAGC) on a MiSeq following the manufacturer’s guidelines.
Miseq dna library preparation protocol
Manufactured by Illumina
The MiSeq DNA library preparation protocol is a laboratory procedure used to prepare DNA samples for sequencing on the MiSeq benchtop sequencing system. The protocol involves several steps to generate sequencing-ready libraries from DNA samples.
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Lab products found in correlation
2 protocols using miseq dna library preparation protocol
1
Amplification and Sequencing of 16S rRNA
2
16S rRNA Gene Amplification and Sequencing
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The V3-V4 region of the 16S rRNA gene was ampli ed using PCR primers (341F 5'-CCTACGGGNGGCWGCAG-3'; 805R 5'-GACTACHVGGGTATCTAATCC-3'). Ampli cation was performed using the HotStarTaq Plus Master Mix Kit (Qiagen, USA) under the following conditions: 94°C for 3 min, followed by 28 cycles of 94°C for 30 s, 53°C for 40 s and 72°C for 1 min, with a nal elongation step at 72°C for 5 min. PCR products were con rmed using a 2% agarose gel. PCR products were then used to prepare DNA libraries following Illumina MiSeq DNA library preparation protocol. Sequencing was performed at the University of Arizona Genetics Core (UAGC) on a MiSeq following the manufacturer's guidelines. All sequence data were deposited in GenBank under Sequence Read Archive number PRJNA810064.
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