Bacterial genomic DNA was prepared using a DNeasy bacterial DNA extraction kit (QIAGEN, Hilden, Germany) following the vendor’s recommendation. S. Typhi ISP2825 (GenBank number CP080960), a drug-susceptible clinical isolate, was used as a negative control for PCR reactions [39 (link)]. The PCR primer sequences and reaction conditions used are summarized in S4 Table . Green Taq DNA polymerase with provided buffers (GenScript, USA Inc., Piscataway, NJ, USA) was used for pltB, blaTEM1, dhfR7, sul1, catA1, parC, parE, blaCTX-M-15, macA, acrB, and acrR. Phusion high fidelity DNA polymerase with the provided GC buffer (New England BioLabs, Ipswich, MA, USA) was used for gyrA, gyrB, and qnrS. PCR reaction steps were: pre-denaturation at 95°C for 3 min, 34 cycles of denaturation at 95°C for 30 sec, annealing (see S4 Table ), and extension at 72°C for 1 min/kb (see S4 Table for amplicon size), and final extension at 72°C for 7 min using a C1000 Touch Thermal Cycle (BIO-RAD, Hercules, CA, USA). PCR results were run on 1% agarose gels, along with GeneRuler 1 kb plus DNA ladder (ThermoFisher Scientific, catalog # SM1333, USA), and imaged using an iBright CL1500 Imaging system (ThermoFisher Scientific, Waltham, MA, USA).
Green taq dna polymerase
Manufactured by GenScript
Sourced in United States
Green Taq DNA polymerase is a thermostable DNA polymerase derived from the bacterium Thermus aquaticus. It is commonly used in the polymerase chain reaction (PCR) technique for DNA amplification. The enzyme possesses 5' to 3' DNA polymerase activity and is suitable for a wide range of PCR applications.
Automatically generated - may contain errors
Lab products found in correlation
C1000 touch thermal cycle, by Bio-Rad (2 mentions)
Dneasy bacterial dna extraction kit, by Qiagen (2 mentions)
Ibright cl1500 imaging system, by Thermo Fisher Scientific (2 mentions)
Generuler 1 kb plus dna ladder, by Thermo Fisher Scientific (1 mentions)
Phusion high fidelity dna polymerase, by New England Biolabs (1 mentions)
T100 thermal cycler, by Bio-Rad (1 mentions)
Amersham typhoon scanner, by Cytiva (1 mentions)
Sybr gold, by Thermo Fisher Scientific (1 mentions)
Evagreen dye, by Biotium (1 mentions)
Mj opticon 2, by Bio-Rad (1 mentions)
Revertaid h minus reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Herculase 2 fusion dna polymerase, by Agilent Technologies (1 mentions)
6 protocols using green taq dna polymerase
1
Bacterial DNA Extraction and PCR Amplification
2
Sensitive Salmonella Detection by PCR
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PCR amplification was performed in 1× PCR buffer containing 1·5 mmol l−1 MgCl2, 2·5 U Green Taq DNA polymerase (Genscript, Piscataway, NJ), 0·2 mmol l−1 dNTP and primers oriCF (5′- TTATTAGGATCGCGCCAGGC-3′) and oriCR (5′- AAAGAATAACCGTTGTTCAC-3′) which detect all Salmonella at a final concentration of 0·5 μmol l−1 and combined with 5 μl of extracted DNA in a reaction volume of 50 μl in a T100™ Thermal Cycler (Bio-Rad laboratories, Hercules, CA) (Widjojoatmodjo et al. 1991 (link); Levy et al. 2008 (link)). Cycling parameters were as follows: 94°C for 3 min, followed by 30 cycles at 94°C for 30 s, 55°C for 30 s and 72°C for 30 s with a final extension of 72°C for 5 min. The primers described above yielded an amplicon size of 163 bp. PCR products were separated on 2% (w/v) agarose gels, stained with ethidium bromide and visualized using a UV transilluminator.
3
Telomerase Activity Measurement Assay
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Telomerase activity measurement was performed in 25 µL reactions. Each reaction was prepared using 5x TRAP buffer (100 mM Tris-HCl pH 8.3, 7.5 mM MgCl2, 315 mM KCl, 0.025% Tween-20, 5 mM EGTA, 0.5 mg/mL BSA), TRAP primer mix (ACX primer 10 µM, NT primer 10 µM, TSNT 100 pM), TS primer (10 µM), dNTP mix (10 mM each), 1 units of Green Taq DNA polymerase (GenScript), 2 µL of the sample (anti-FLAG immunoprecipitate) and 2 µL of either DMSO (final concentration of 0.25% v/v) or BIBR1532 (Selleck Biochem). Ten µL of TRAP reactions were later loaded onto a 10% (w/v) 19:1 acrylamide:bis, non-denaturing gel and subjected to electrophoresis for 90 min at 400 V. Telomerase products were visualized by gel staining with a 100 mL solution of 1X SYBR Gold (Invitrogen) for 30 min. The image acquisitions were made using an Amersham Typhoon scanner (cytiva life sciences).
4
Quantitative Analysis of Gene Expression
5
Genotyping rs2241766 Polymorphism by PCR-RFLP
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A phenol-chloroform extraction (Barker et al., 1998) was used to isolate the genomic DNA from the peripheral blood samples, followed by precipitation in absolute ethanol. The DNA pellet was resuspended in Tris-EDTA (pH 7.8) at a final concentration of 0.1-1.0 μg/μl and stored at -20 °C until used for genetic analysis. The rs2241766 polymorphism was genotyped through PCR-RFLP using specific oligonucleotide primers and a MJ Mini PTC1148 thermocycler (Bio-Rad, Hercules, CA, USA) (Mackevics et al., 2006) (link). The PCR included 200 ng of genomic DNA and 0.5 μm of each of primer (forward 5´-TGT GTG TGT GGG GTC TGT CT-3´and reverse 5´-TGT GAT GAA AGA GGC CAG AA-3´; IDT, Coralville, IA, USA), 0.2 mmol dNTPs, 1.5 mmol MgCl 2 , and 1 unit of Green Taq DNA Polymerase (GenScript, Nueva Jersey, USA). The amplification program consisted of 35 cycles at 95 °C/45 s, 58 °C/45 s, and 72 °C/45 s. The amplicons, of 305 bp in length, were digested with the AvaI restriction enzyme (New England Biolabs, Ipswich, MA, USA), loaded in a 2% agarose gel prior to electrophoresis, stained with ethidium bromide, and visualized in a UVP m2UV High Performance Transilluminator (Upland, CA, USA). The expected results were two fragments (i.e., 204 bp and 101 bp) for the mutant G allele and a single 305 bp fragment (i.e., non-digested) for the wildtype T allele.
6
Bacterial Antibiotic Resistance Profiling
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Bacterial genomic DNA was prepared using a DNeasy bacterial DNA extraction kit (QIAGEN) following the vendor's recommendation. The PCR primer sequences and reaction conditions used are summarized in Table 3. Green Taq DNA polymerase with provided buffers (GenScript) was used for pltB, blaTEM1, dhfR7, sul1, catA1, parC, parE, blaCTXM15, macA, acrB, and acrR. Phusion high fidelity DNA polymerase with the provided GC buffer (New England BioLabs) or Herculase II fusion DNA polymerase (Agilent) was used for gyrA, gyrB, and qnrS.
PCR reaction steps were: pre-denaturation at 95℃ for 3min, 34 cycles of denaturation at 95℃ for 30 sec, annealing (see Table 3), and extension at 72℃ for 1 kb/min (see Table 3 for amplicon size), and final extension at 72℃ for 7 min using a C1000 Touch Thermal Cycle (BIO-RAD).
PCR results were run on 1% agarose gels and imaged using an iBright CL1500 Imaging system (ThermoFisher Scientific).
PCR reaction steps were: pre-denaturation at 95℃ for 3min, 34 cycles of denaturation at 95℃ for 30 sec, annealing (see Table 3), and extension at 72℃ for 1 kb/min (see Table 3 for amplicon size), and final extension at 72℃ for 7 min using a C1000 Touch Thermal Cycle (BIO-RAD).
PCR results were run on 1% agarose gels and imaged using an iBright CL1500 Imaging system (ThermoFisher Scientific).
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