Total RNA was extracted using an RNeasy Mini Kit (QIAGEN, Maryland, USA) and reverse-transcribed into cDNA with SuperScript III First-Strand Synthesis SuperMix (Invitrogen, California, USA) according to the manufacturer's instructions. Quantitative PCR was performed by following the instructions of the HotStarTaq Master Mix Kit (QIAGEN). The reactions were performed in accordance with the following conditions: 95°C for 15 min, 25 cycles of 95°C for 15 s, 58°C for 30 s, and 72°C for 30 s, followed by 72°C for 10 min. The RT-PCR primers used were as follows:
Hotstartaq master mix kit
Manufactured by Qiagen
Sourced in Germany, United States, United Kingdom
The HotStarTaq Master Mix Kit is a ready-to-use PCR (Polymerase Chain Reaction) reagent that contains all the necessary components for performing PCR amplification. It includes a thermostable HotStarTaq DNA Polymerase, optimized buffer, and dNTPs.
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Lab products found in correlation
Ez dna methylation gold kit, by Zymo Research (6 mentions)
Trizol reagent, by Thermo Fisher Scientific (6 mentions)
Qiaamp dna mini kit, by Qiagen (5 mentions)
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (5 mentions)
Ez dna methylation kit, by Zymo Research (4 mentions)
Qiaquick gel extraction kit, by Qiagen (4 mentions)
Rnalater, by Qiagen (4 mentions)
C1000 thermal cycler, by Bio-Rad (4 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
Hotstartaq master mix, by Qiagen (3 mentions)
Alexa fluor 488 goat anti rabbit igg, by Cell Signaling Technology (3 mentions)
Alexa fluor 555 goat anti mouse igg, by Cell Signaling Technology (3 mentions)
Pyromark q96 id, by Qiagen (3 mentions)
Superscript 3 first strand synthesis supermix, by Thermo Fisher Scientific (2 mentions)
Alphaimager hp, by Bio-Techne (2 mentions)
Cyanine5 maleimide, by Lumiprobe (2 mentions)
Qiaspin miniprep kit, by Qiagen (2 mentions)
Megascript t7 high yield transcription kit, by Thermo Fisher Scientific (2 mentions)
Rotorgene corbett 6000, by Qiagen (2 mentions)
Dneasy tissue kit, by Qiagen (2 mentions)
137 protocols using hotstartaq master mix kit
1
Extracting and Quantifying RNA Expression
2
Pyrosequencing of DPYSL2, NFATC1, miR3138
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Design of primers for DPYSL2, NFATC1, and miR3138 and preparation of the pyrosequencing amplicons were done at the University of Michigan Epigenomics Core using a published protocol [86 ]. Briefly, 20 ng of bisulfite-treated DNA was used for PCR amplification (45 cycles) using HotStarTaq Master Mix Kit (203443; Qiagen) and 200 pM of amplicon-specific primers (Supplemental Table S7). For DYSPL2, 5% DMSO was added to the PCR reaction. To verify the expected size of the amplicons, 5 μl of each PCR reaction was run on an ethidium bromide-agarose gel. The amplicons were submitted to the University of Michigan Sequencing Core for sample preparation and pyrosequencing on the PyroMark Q96 MD [87 ].
3
Characterizing Mutant Growth in M9 Media
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Isolated mutants were tested for growth over the course of 1 week (Appendix Table S3 ). Individual colonies were isolated on LB agar plates and used to inoculate pre‐cultures grown overnight in 2 ml of glucose M9 minimal liquid media in 10‐ml tubes. The following morning, pre‐cultures were pelleted at 2,000 g and gently resuspended (by pipetting) in M9 minimal medium without a carbon source and this spinning and resuspension was repeated twice to wash the cells of residual glucose. The final resuspension was in 2 ml of M9 minimal medium without a carbon source. The growth test tubes consisting of 2 ml of M9 minimal medium plus the corresponding innovative carbon source were inoculated with the washed cells at a dilution factor of 1:200. Growth was monitored over the course of 1 week by visually inspecting for increased cellular density, noting that the cultures had become opaque from cell growth. Once growth was observed, colony PCR was conducted (Qiagen HotStarTaq Master Mix Kit) with the primer sequences listed in Appendix Table S5 . DNA sequencing of PCR products was conducted by Eton Bioscience Inc using their SeqRegular services. DNA sequencing was utilized to confirm the designed mutations were as expected and to confirm that no other mutations had been acquired in the regions of interest during the growth test.
4
PCR-based identification of mlr gene cluster
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For the identification of the mlr gene cluster, a PCR using the specific primer sets MF/MR [22 (link)], mlrBf1/mlrBr1, mlrCf1/mlrCr1 and mlrDf1/mlrDr1 [25 (link)] was carried out. Amplifications were performed in 50 µL of total volume containing final concentration of 0.25 µM of each primer, 1× PCR buffer (HotStarTaq Master Mix kit, QIAGEN, Hilden, Germany) and 1 µL of DNA. Amplification of the mlrA gene in a PCR thermal cycler (Techne TC-5000, Bibby Scientific, Staffordshire, UK) was performed under the following conditions: initial activation step at 95 °C for 15 min, followed by 35 cycles of 94 °C for 30 s, 57 °C for 40 s and 72 °C for 40 s. Final extension step at 72 °C for 10 min. Amplification of mlrB, mlrC and mlrD genes were performed with an initial activation step at 95 °C for 15 min, followed by 40 cycles of 95 °C for 30 s, 60 °C for 40 s and 72 °C for 40 s. Final extension step was set at 72 °C for 10 min. PCR products were separated on 1.5% agarose gel electrophoresis and visualized on a AlphaImager HP (Alpha Innotech, San Leandro, CA, USA).
5
Plasmid DNA Preparation and mRNA Synthesis
6
ARMS PCR for JAK2 and MPL Mutations
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The ARMS PCR testing for JAK2 V617F mutation [4 (link)] and MPL W515L/K mutations [16 ] was performed as described. The assay for JAK2 V617F identification utilized a pair of outer primers as well as wild-type/mutant specific primers to amplify the wild-type or mutant fragment with an internal amplification control sequence in a single reaction. Detection of MPL W515L/K mutations was carried out in a single PCR reaction with 2 outer primers designed to amplify the internal amplification control flanking the mutation site. The 2 specific primers for either the wild-type or the mutant sequence were employed to distinguish the wild-type allele from the mutant one by fragment size. A similar assay was also developed to detect MPL W515K mutation with the other mutation specific primer. All the primers used in the ARMS method are listed in Table 1 . Amplifications were performed for 35 cycles with the HotStarTaq Master Mix Kit (Qiagen).
7
PCR-based DNA Variant Detection
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Genomic DNA was amplified by PCR using primers forward, 5′-AGACTGCGAGATGGGAGAAG-3′, and reverse, 5′-GGTAGAGACGCAGAGCCAAG-3′, and the HotStar Taq Master Mix kit (QIAGEN). The PCR product was digested with AleI and analyzed by gel electrophoresis on a 2% agarose gel. For the SNP variant C, DNA digestion results in two bands of 279 and 129 bp.
8
Leptospira Serogroup and Genotyping
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First, serogroups of the isolates were determined through the MAT using a panel of eight polyclonal anti-sera against the eight serovars reported in Section 4.2 . The agglutination with specific antiserum was used to identify the presumptive strain’s serogroup [84 ].
Isolated Leptospira were genotyped using a multilocus sequence typing (MLST) scheme based on housekeeping genes [87 (link),88 (link),89 (link)].
Moreover, the Leptospira species were identified from positive pathogenic and intermediate Leptospira PCR reactions, using primer for rrs2 gene and 16S rRNA gene, respectively [86 (link),88 (link)].
The amplification of each target gene was realized with HotStarTaq Master Mix Kit (Qiagen, Hilden, Germany), and further sequenced (BMR Genomics, Padova, Italy) using the same amplification primer sets and analyzed using BioEdit Software [90 ]. Phylogenetic analysis was performed by the maximum likelihood method based on the Tamura–Nei model using MEGA 10 software [91 (link)].
Isolated Leptospira were genotyped using a multilocus sequence typing (MLST) scheme based on housekeeping genes [87 (link),88 (link),89 (link)].
Moreover, the Leptospira species were identified from positive pathogenic and intermediate Leptospira PCR reactions, using primer for rrs2 gene and 16S rRNA gene, respectively [86 (link),88 (link)].
The amplification of each target gene was realized with HotStarTaq Master Mix Kit (Qiagen, Hilden, Germany), and further sequenced (BMR Genomics, Padova, Italy) using the same amplification primer sets and analyzed using BioEdit Software [90 ]. Phylogenetic analysis was performed by the maximum likelihood method based on the Tamura–Nei model using MEGA 10 software [91 (link)].
9
Leptospira Detection and Identification
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DNA was extracted from each tissue sample using the Quick-DNA Plus Kit (Zymo Research, Irvine, CA, USA) following the manufacturer’s instructions. All samples were tested by multiplex Real-Time PCR, identifying the Leptospira genus-specific target located on 16S rRNA gene and the specific target for pathogenic species (lipL32 gene) [32 (link),33 (link)]. The Real-Time PCR assay was performed on a Rotorgene Corbett 6000 (Corbett Research, Sidney, Australia) with the following thermal conditions: a holding stage of 95°C for 5 min, 45 cycles of 95°C for 15 sec, and 60°C for 30 sec. Samples were considered positive with a Ct < 40.
Finally, from PCR-positive tissue samples, Leptospira species were identified using a primer for the rrs2 gene [34 (link)]. Amplification of each target gene was carried out using a HotStarTaq Master Mix Kit (Qiagen, Hilden, Germany). Amplicons were further sequenced (BMR Genomics, Padova, Italy) using the same amplification primer sets and analyzed using BioEdit Software [35 ]. Phylogenetic analysis based on the rrs2 gene was performed using the Maximum Likelihood method based on the Tamura-Nei model using MEGA 10 software [36 (link)].
Finally, from PCR-positive tissue samples, Leptospira species were identified using a primer for the rrs2 gene [34 (link)]. Amplification of each target gene was carried out using a HotStarTaq Master Mix Kit (Qiagen, Hilden, Germany). Amplicons were further sequenced (BMR Genomics, Padova, Italy) using the same amplification primer sets and analyzed using BioEdit Software [35 ]. Phylogenetic analysis based on the rrs2 gene was performed using the Maximum Likelihood method based on the Tamura-Nei model using MEGA 10 software [36 (link)].
10
Bacterial Colony Screening via PCR
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Example 8
Bacterial colonies were screened for the presence of vectors containing the desired sequences via colony PCR using the HotStarTaq Master Mix Kit (Qiagen; product #203445) and the appropriate forward and reverse primers (Appendix 1). Selected colonies were lightly touched with a 20 μl pipette tip and touched briefly in 2 ml LB for small scale culture, and then resuspended in the PCR mix. PCR was performed with a TGradient Thermocycler 96 using a 35-cycle program: denaturation at 95° C. for 15 min; 35 cycles of 94° C. for 30 sec, 55° C. for 30 sec and 72° C. for 2 min; followed by a final extension step of 10 min at 72° C. If appropriate, the PCR mixtures were stored at 4° C. until analysis by agarose gel electrophoresis.
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