Total RNA was extracted using the NucleoSpin® RNA extraction kit (Macherey-Nagel, Germany), as recommended by the manufacturer. Total RNAs were reverse transcripted using the iScript cDNA Synthesis Kit (Bio-Rad, USA). Genes of interest were amplified using 5x FIREPol® Master Mix (Solis BioDyne, Estonia). Primer sequences for hTERT, VEGF and GAPDH amplification were: hTERT forward primer 5’-TGAACTTGCGGAAGACAGTGG-3’ , hTERT reverse primer 5’-ATGCGTGAAACCTGTACGCCT-3’ , VEGF forward primer 5’-GGAGGGCAGAATCATCACGAAG-3’ , VEGF reverse primer 5’-CACACAGGATGGCTTGAAGATG-3’ , GAPDH forward primer 5’-TGGGATGGACTGTGGTCATGAG-3’ , GAPDH reverse primer 5’-ACTGGCGTCTTCACCACCATGG-3’ . Reactions were initiated at 95°C for 3 min, followed by 94°C, 58°C (for hTERT), or 60°C (for VEGF and GAPDH) for 30 s, and 70°C for 30 s, 40 cycles for hTERT, or 35 cycles for VEGF and GAPDH, then followed by an elongation step at 72°C for 7 min. After amplification, PCR products were separated on 2% agarose gels and visualized by SYBR® Safe DNA Gel Stain (Invitrogen, Life Technologies, USA).
Firepol master mix
Manufactured by Solis BioDyne
Sourced in Estonia
FIREPol Master Mix is a ready-to-use solution for conducting polymerase chain reaction (PCR) experiments. It contains a DNA polymerase enzyme, necessary buffers, and reagents for the amplification of DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
3730xl dna analyzer, by Thermo Fisher Scientific (6 mentions)
Abi 7500 real time pcr system, by Thermo Fisher Scientific (6 mentions)
Geneamp pcr system 9700, by Thermo Fisher Scientific (4 mentions)
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (4 mentions)
Bigdye terminator v3.1 cycle sequencing, by Thermo Fisher Scientific (3 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Proflex pcr system, by Thermo Fisher Scientific (2 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Nucleospin rna extraction kit, by Macherey-Nagel (1 mentions)
Sybr safe dna gel stain, by Thermo Fisher Scientific (1 mentions)
Miseq platform, by Illumina (1 mentions)
Agencourt ampure xp magnetic beads, by Beckman Coulter (1 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Agilent 2100, by Agilent Technologies (1 mentions)
Nucleospin tissue dna extraction kit, by Macherey-Nagel (1 mentions)
3 race system, by Thermo Fisher Scientific (1 mentions)
Firescript rt cdna synthesis mix, by Solis BioDyne (1 mentions)
Sybr gold stain, by Thermo Fisher Scientific (1 mentions)
T100 thermocycler, by Bio-Rad (1 mentions)
45 protocols using firepol master mix
1
Gene Expression Analysis in Cells
2
16S rDNA Sequencing of Gut Microbiome
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Total DNA from the stool samples was extracted using the ZymoBiomics DNA/RNA mini kit (ZymoResearch Scientific, Irvine, CA, USA) in accordance with the manufacturer’s protocol. DNA from each sample was amplified using specific primers targeting the V1–V3 region of 16S rDNA [37 (link)]. PCR reaction contained 1 ng of DNA, 5xFIREPol MasterMix (Solis BioDyne, Tartu, Estonia) and 2 µM of each primer (10 µM). The reaction conditions for PCR amplification were 95 °C for 15 min; 25 cycles of 95 °C for 20 s, 56 °C for 30 s and 72 °C for 1 min; and final elongation at 72 °C for 5 min. The products of amplification were verified by agar electrophoresis. DNA libraries for Illumina sequencing were prepared using the index PCR reaction with input of 1 ng of DNA. The reaction conditions for the PCR were 95 °C for 15 min; 12 cycles of 95 °C for 10 s, 55 °C for 30 s and 72 °C for 90 s; and final elongation at 72 °C for 5 min. Index PCR amplification products were purified using 1.8x Agencourt AMPure XP magnetic beads (BeckmanCoulter, Brea, CA, USA). DNA libraries were validated by Agilent 2100 (Agilent Technologies, Santa Clara, CA, USA) and quantified by Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). Mixed amplicons were pooled and sequenced using an Illumina MiSeq platform via a 300 bp paired-end reads Illumina sequencing system (Illumina, San Diego, CA, USA).
3
Amplification of Amylase Gene via PCR
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From extracted DNA, the amylase-coding gene was amplified using PCR (polymerase chain reaction). This was done using the following specific primer-forward: 5′AGTGCTGAA ACGGCGAACAAATCGAA3′and reverse: 5′CTCAATGGGGA AGA GAA CCGCTTAAG 3'. The primers were used by Prasad [29 ] with few modifications. The PCR mixture was prepared using Solis BioDyne 5x FIREPol® Master Mix ready to load. 1 µL of template DNA and 1 µL of each primer were added right before loading the sample in the PCR machine. Reaction set up for PCR was carried out for 40 µL reaction volume in a 0.2 mL thin-walled PCR tube.
The thermal cycle was programmed as follows: initial denaturation at 94°C for 2 min; 30 cycles of denaturation at 94°C for 30 sec, annealing at 50°C for 30 sec, extension at 72°C for 2 min, and a final extension at 72°C for 5 min. The PCR was carried out for 30 cycles. Afterwards, the amplicon was stored at −20°C for further work [30 (link)].
The thermal cycle was programmed as follows: initial denaturation at 94°C for 2 min; 30 cycles of denaturation at 94°C for 30 sec, annealing at 50°C for 30 sec, extension at 72°C for 2 min, and a final extension at 72°C for 5 min. The PCR was carried out for 30 cycles. Afterwards, the amplicon was stored at −20°C for further work [30 (link)].
4
ExPEC and UPEC Genotyping in E. coli
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Phylogroups and ExPEC VG carriage of the 104 E. coli were identified using PCR-based methods. Genomic DNA was extracted using Nucleospin® Tissue DNA extraction kits (Machery-Nagel, Germany). Clermont E. coli phylo-typing, which consists of a quadruplex PCR panel and two simplex PCRs, was performed to differentiate E. coli phylogroup A, B1, B2, C, D, E, F and E. coli cryptic clades35 (link). VGs associated with extraintestinal pathogenesis of E. coli, including adhesion (afa, crl, fimA, iha, papC, sfa/foc, yfcV, and tsh), cytotoxicity (hlyA, hlyE, cnf, sat, and vat), iron acquisition (iroN, fyuA, irp1, chuA, iutA, and iucD), and bacterial cell protection (bssS, hmsP, iss, and kpsII) were detected by PCR (see Supplementary Table S4 online). All PCRs were conducted in a 25 µL total volume containing 5X Firepol Master Mix (Solis Biodyne, Estonia), and the concentration of each primer was 0.2 µM.
Isolates that tested positive for ≥ 2 out of 5 VGs (papC, sfa/foc, afa, iutA, and kpsII) were presumably classified as ExPEC, whereas isolates demonstrating ≥ 3 out of 4 VGs (yfcV, vat, fyuA, and chuA) were presumably classified as UPEC4 (link),6 (link).
Isolates that tested positive for ≥ 2 out of 5 VGs (papC, sfa/foc, afa, iutA, and kpsII) were presumably classified as ExPEC, whereas isolates demonstrating ≥ 3 out of 4 VGs (yfcV, vat, fyuA, and chuA) were presumably classified as UPEC4 (link),6 (link).
5
3' RACE Protocol for Identifying mRNA Transcripts
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Total RNA used for the 3′ RACE experiment was extracted from embryos prepared from wild-type animals as described above. The 3′ RACE System from Invitrogen™ (Catalog number: 18373019) was used for cDNA synthesis according to the kit protocol with 2.5 μg total RNA per reaction. To control RNA and primer quality, cDNA was also synthesized using the FIREScript® RT cDNA synthesis MIX with Oligo (dT) primers from Solis BioDyne. PCR reactions were set up using the 5X FIREPol® Master Mix (Solis BioDyne). Each reaction was set up identically using 2 μl cDNA, 1 μl gene specific primer (10 μM) and 1 μl universal adapter primer provided from the Invitrogen™ 3′ RACE System kit in a total reaction volume of 50 μl. The gene specific cfp-1 primers used were: cfp-1 F1 and cfp-1 F2 (Supplementary Table S2 ). For the Oligo (dT) primed cDNA controls cfp-1 R1 or cfp-1 R2 (Supplementary Table S2 ) were used instead of the universal adapter primer from the RACE kit. PCR products were separated on a 1.5% agarose TAE (Tris–acetate–EDTA) gel and individual bands were cut out, purified and sequenced.
6
Genotyping CCR5 Polymorphism Using ASO-PCR
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Genotyping of the CCR5 polymorphism was carried out using the ASO-PCR technique. For the Δ32 mutation, genotyping of each sample was carried out in a 25 μL reaction medium containing 15 µL of pure water (molecular biology grade water), 04 µL 5X FIREPOL®Master Mix (Solis BioDyne) 5x, 0.5 µL of each primer (0.5 µM), and 5 µL of DNA (10 ng/µL). Table 1 shows the primer pairs used (Guleria et al., 2012). The thermocycling parameters were an initial denaturation at 94°C for 05 min followed by 50 amplification cycles, then 40 amplification cycles (denaturation at 94°C, 30 s, hybridisation at 55°C for 30 s, and elongation at 72°C, 30 s), and final elongation at 72°C, 7 min using the Gene Amp®PCR System 9700 (Applied Biosystems).
The PCR products were then electrophoresed on a 2% agarose gel, migrated for 45 min (100 volts), and visualised using the “Vilber” apparatus (Vilber Lourmat).
The PCR products were then electrophoresed on a 2% agarose gel, migrated for 45 min (100 volts), and visualised using the “Vilber” apparatus (Vilber Lourmat).
7
PCR Amplification and Enzymatic Digestion
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A reaction mixture with a total volume of 25 µL (per sample) composed of 4 µL of 5X FIREPOL®Master Mix (Solis BioDyne), 0.5 µL of each primer (0.5 µM), 14 µL of sterile water, and 5 µL of DNA (10 ng/µL). The sequences of the primer pairs used are recorded in Table 1 (Guleria et al., 2012; Tiofack et al., 2020). Amplification was carried out using the Gene Amp®PCR System 9700 thermal cycler (Applied Biosystems) following the amplification programme: initial denaturation at 94°C for 5 min followed by 40 amplification cycles (denaturation at 94°C, 30 s, hybridisation at 57°C for 30 s, and elongation at 72°C, 30 s), and final elongation at 72°C for 7 min. The PCR products were then enzymatically digested with the MboII enzyme at 37°C for 3 h. Finally, the digestion products were subjected to electrophoretic migration for 45 min (100 V) on a 2% agarose gel containing 2 µL of ethidium bromide (10 mg/mL) and visualised using the “Vilber” apparatus (vilber Lourmat).
8
Oligonucleotide Library Formation and Thrombin Binding Assay
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Both the forward and reverse primers of sequences, 5′ATC AGT TCG AGC AGA TGA GC′3 and 5′CCA GAC TGC GAG CGT TTT TTT TTT-3′ respectively as well as a 10–100 nt oligonucleotide ladder, were purchased from IDT. Candidate polynucleotide sequences were also purchased from IDT using their Ultramer® technology including two scrambled sequences (SC01 and SC02 ), which correspond to the randomised sequence of the longest polynucleotide sequences tested. Terminal deoxynucleotidyl transferase (TdT), dNTPs, and human thrombin were purchased from Sigma Aldrich. SYBR gold stain was purchased from Thermo Scientific. 5x FIREPol® Master Mix and sample loading buffer were purchased from Solis Biodyne. The Buffer SB1: 50 mM Tris–HCl, 250 mM KCl and 7.5 mM MgCl2 pH 7.4 (5×) were used for all TdT catalysed library formation reactions and library selection steps. All oligonucleotide purification steps were performed using an oligonucleotide purification kit from Norgen Biotek. Purification of the PCR products was performed using a PCR clean-up kit purchased from Macherey-Nagel GmbH. All electrophoretic mobility shift assays (EMSA); (5–6%) and agarose gels (2%) were prepared in house. All PCR reactions were prepared in a dedicated laminar flow cabinet and all PCR experiments were performed on a T100 thermocycler (BioRad).
9
RNA Isolation and RT-qPCR Analysis
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For RNA isolation, cells were lysed directly in Trizol (Invitrogen) reagent. RNA was isolated according to the manufacturer’s instructions. 1 μg of total RNA was used for cDNA synthesis using oligo (dT) primers and Superscript III first strand kit (Invitrogen). For RT-PCR analysis, cDNA was amplified using 5x FIREPol Master Mix (Solis BioDyne, #04-11-00125). For RT-qPCR analysis, cDNA amplification was performed using SensiFast Sybr HiROX kit (Bioline, #Bio-92020). Primer information was provided in the Supplementary Table S2 .
10
Germline Transmission of mCherry in Chickens
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In order to test the capability of mCherry CB PGCs to colonize the gonads and go germline after injection into H&H 14 embryos we injected 3000 cells per embryo and raised the resulting male embryos to sexual maturity like described before7 (link). Semen was collected by abdominal massage and isolated gDNA was tested with primers specific for mCherry. For detection of mCherry gene, the following primers were used: 5′-CAGGACGGCGAGTTCATCTACAAG-3′ and 5′-CAGCTTCAGCCTCTGCTTGATCTC-3′. 5x FIREPol MasterMix (Solis BioDyne) was used for all PCRs according to manufacturer’s protocol. Roosters with mCherry positive semen were bred to white leghorn hens and the resulting eggs were icubated at 37°C. On embryonic day seven, several eggs were cracked and mCherry positive embryos were visualized by using a Leica stereomicroscope.
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