HotStarTaq DNA Polymerase (25000)

Protocol tips

Upstream tips	Protocol tips	Downstream tips

Publication protocol

PCR amplification and Nanopore library construction and sequencing: Primer sets 16S27F/16SR1495 (5’-AGAGTTTGATCMTGGCTCAG-3’ / 5’- TACGGYTAC CTTGTTACGACTT-3’) [34] and Cn_pPer11F/Cn_pPer986R (5’-CCTCGACCACCGCC GCGA-3’ / 5’-CGGCTCCTGCCCCTCGGG-3’) fused to Nanopore-specific oligonucleo- tides were used for 16S rRNA and purine permease amplifications, respectively. PCR amplifica- tions of all replicates of the mock bacterial communities (Cn-spiked or not) and corn leaf samples (diseased and healthy) were performed using HotStar Taq Plus DNA polymerase kit (Qiagen, Canada) and barcoded using the SQK-PBK004 PCR Rapid Barcoding kit (ONT) targeting 16S rRNA and purine permease genes where required. Briefly, a PCR reaction (total volume of 60 μl) consisted of 6 μl QIAGen HotStar 10x Buffer, 12 μl 5x Q-sol, 0.75 μl of 2.5 mM dNTPs, 0.6 μl 20 nM of each primer, 1.8 μl of LWB Barcodes (ONT), 0.6 μl of Taq polymerase plus (5U/μl; Qiagen), 6 μl DNA template (5 ng/μl), and 21.65 μl of PCR H2O. PCR parameters performed in a TProfe- sional thermocycler (Biometra, Germany) were an initial denaturation at 95 ̊C, 5 min; followed by 40 cyles of 95 ̊C, 30 sec, 65 ̊C, 30 sec, 72 ̊C, 60 sec; and a final extension at 72 ̊C, 5 min. For quality control, 3 μl of the PCR amplicons were verified by agarose (1.3%) gel electrophoresis. The remaining 57 μl of PCR products were purified by Amicon Ultra-0.5 Centrifugal Filters (Milli- pore, Canada). The DNA concentration of the purified PCR products were quantified by Qubit High Sensitivity DNA assay (ThermoFisher, Canada) as recommended by the manufacturer.

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing PCR Hot start PCR - Bacterial DNA using HotStarTaq DNA Polymerase (25000) from Qiagen.

Manufacturer protocol

Download the product protocol from Qiagen for HotStarTaq DNA Polymerase (25000) below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing PCR Hot start PCR - Bacterial DNA using HotStarTaq DNA Polymerase (25000) from Qiagen. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.