Kapa Biosystems HIFI HOTSTART READY MIX

PCR Hot start PCR - Bacterial DNA

Experiment
PCR Hot start PCR - Bacterial DNA
Product
Kapa Biosystems HIFI HOTSTART READY MIX from Fisher Scientific
Manufacturer
Fisher Scientific

Protocol tips

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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Discussion

Discussion

5 years ago

Author: Qatar

When should I add the polymerase for hot start PCR?

I keep getting a non-specific band in PCR so I would like to try a hot start PCR manually. How should I prepare the mix and at which step should I be adding the polymerase?

Discussion

5 years ago

Author: Janina Reinhardt Switzerland

Hot start PCR using phusion green DNA polymerase

I am currently performing hot start PCR using phusion green hot start DNA polymerase in order to amplify a 8.8kb insert. However, I cannot get any amplified product at all. I have tried changing some parameters but I am not sure what to do. Any help?

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Papers

Check out relevant papers found by Labettor's AI that are relevant for performing PCR Hot start PCR - Bacterial DNA using Kapa Biosystems HIFI HOTSTART READY MIX from Fisher Scientific.

Paper title
Comprehensive detection and identification of bacterial DNA in the blood of patients with sepsis and healthy volunteers using next-generation sequencing method - the observation of DNAemia
Full paper
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Manufacturer protocol

Download the product protocol from Fisher Scientific for Kapa Biosystems HIFI HOTSTART READY MIX below.

Download PDF Download manufacturer protocol

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