Kapa Biosystems HIFI HOTSTART READY MIX

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Amplicon size should be consistent for each target sequence and limited to approximately 65 - 100 bp.

Upstream tips
Amplicon size should be consistent for each target sequence and limited to approximately 65 - 100 bp.

Publication protocol

DNA isolation, quantitation and quantification

Microbial DNA was isolated from 1.5-ml of blood sample according to the method described by Gosiewski et al. with the employment of a ready-to-use Blood Mini kit (A&A Biotechnology) [].

16S Library preparation and sequencing

The library preparation procedure followed the 16S Metagenomic Sequencing Library Preparation Protocol - Preparing 16S Ribosomal RNA Gene Amplicons for the Illumina MiSeq System (Illumina). DNA amplification was performed with the use of KAPA HiFi HotStart ReadyMix kit (KAPA BIOSYSTEMS) on C100 thermal cycler (BioRad), as a nested PCR. The content of PCR reaction mix was the following: 1st amplification—DNA template (2.5 μl), each 1 μM external primers (5.0 μl), KAPA HiFi HotStart ReadyMix (12.5 μl); 2nd amplification—DNA amplicon (2.5 μl), each 1 μM internal primers (5.0 μl), KAPA HiFi HotStart ReadyMix (12.5 μl).


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Manufacturer protocol

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