Total RNA and cDNA were prepared respectively by the RNeasy Mini Kit (Cat#74104, QIAGEN) and the RevertAid First Strand cDNA Synthesis Kit (Cat#K1622, Thermo Scientific). The synthesized cDNA was used as a template for PCR amplification of PIP5K1A (Forward primer: AGA AGA TTC CCT GCG TTC ACC, Reverse primer: GAT CTA GAC TAT GGG TGA ACT CTG ACT CTG) and GAPDH (Forward primer: AAC AGC GAC ACC CAC TCC TC, Reverse primer: GGA GGG GAG ATT CAG TGT GGT) by using the Phire Hot Start II DNA Polymerase Kit (Cat#F122S, Thermo Scientific). The PCR product was analyzed by gel electrophoresis in 1% agarose. The signal was captured and documented with the Proxima C16 Phi+ imaging system (Isogen Lifescience). Densitometric quantification was performed by the software ImageJ 1.50i Software (NIH, Baltimore, MD, Unites States) and represented as fold change relative to control and was normalized relative to GAPDH.
Phire hot start 2 dna polymerase kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Phire Hot Start II DNA Polymerase Kit is a high-performance DNA polymerase designed for sensitive and specific PCR applications. It provides robust amplification of target DNA sequences with improved specificity and sensitivity.
Automatically generated - may contain errors
Lab products found in correlation
Rotor gene q, by Qiagen (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Proxima c16 phi imaging system, by Isogen Life Science (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Loading star, by Dyne Bio (1 mentions)
Deoxynucleoside triphosphates dntps, by Promega (1 mentions)
Decalabel dna labeling kit, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Biodyne b nylon membrane, by Pall Corporation (1 mentions)
Quantitect reverse transcription kit, by Qiagen (1 mentions)
Qiaamp dna blood mini kit, by Qiagen (1 mentions)
5 protocols using phire hot start 2 dna polymerase kit
1
Quantitative mRNA Expression Analysis
2
Virulence Gene Detection in Listeria monocytogenes
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Five virulence genes (actA, inlA,
inlB, plcB, and hlyA)
were detected in isolated colonies from Palcam agar plates, using PCR analysis
with the primers listed inTable 1 . The
colonies in Palcam agar plates were suspended in 50 μL of 0.05N NaOH
(Samchun, Gyeonggi, Korea) with 0.25% sodium dodecyl sulfate (SDS). One hundred
microliters of sterile dH2O were added to the suspension, which was
incubated at 99°C for 15 min. For PCR amplification, this mixture (2
μL) was mixed with Phire Hot Start II DNA Polymerase Kit (Thermo Fisher),
mixed Taq DNA polymerase (20 mM Tris-HCl, pH 7.4 at 25°C; 0.1 mM EDTA; 1
mM DTT; 100 mM KCl; 200 μg/mL BSA; and 50% glycerol), 1× reaction
buffer [1.5 mM MgCl2, 200 μM deoxynucleoside triphosphates
(dNTP; Promega Corporation, Madison, USA)], and 0.5 μM each primer. PCR
was performed by Rotor-Gene Q (Qiagen) with initial denaturation at 98°C
for 30 sec, followed by 35 cycles of 98°C for 5 sec, 60°C for 5
sec, and 72°C for 10 sec and a final extension at 72°C for 1 min.
Twenty microliters of reaction PCR products were mixed with 4 μL loading
star (Dyne Bio, Gyeonggi, Korea), followed by electrophoresis analysis with a
1.5% agarose gel.
inlB, plcB, and hlyA)
were detected in isolated colonies from Palcam agar plates, using PCR analysis
with the primers listed in
colonies in Palcam agar plates were suspended in 50 μL of 0.05N NaOH
(Samchun, Gyeonggi, Korea) with 0.25% sodium dodecyl sulfate (SDS). One hundred
microliters of sterile dH2O were added to the suspension, which was
incubated at 99°C for 15 min. For PCR amplification, this mixture (2
μL) was mixed with Phire Hot Start II DNA Polymerase Kit (Thermo Fisher),
mixed Taq DNA polymerase (20 mM Tris-HCl, pH 7.4 at 25°C; 0.1 mM EDTA; 1
mM DTT; 100 mM KCl; 200 μg/mL BSA; and 50% glycerol), 1× reaction
buffer [1.5 mM MgCl2, 200 μM deoxynucleoside triphosphates
(dNTP; Promega Corporation, Madison, USA)], and 0.5 μM each primer. PCR
was performed by Rotor-Gene Q (Qiagen) with initial denaturation at 98°C
for 30 sec, followed by 35 cycles of 98°C for 5 sec, 60°C for 5
sec, and 72°C for 10 sec and a final extension at 72°C for 1 min.
Twenty microliters of reaction PCR products were mixed with 4 μL loading
star (Dyne Bio, Gyeonggi, Korea), followed by electrophoresis analysis with a
1.5% agarose gel.
3
Rapid Molecular Detection of Listeria Virulence
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Isolated colonies of L. monocytogenes isolates were suspended in 50 μL of 0.25% sodium dodecyl sulfate–0.05 N NaOH solution. One hundred microliters of sterile dH2O was added to the suspension, and the mixtures were incubated at 99°C for 15 min. Aliquots (2 μL) of the mixtures were mixed with the components of the Phire Hot Start II DNA Polymerase Kit (Thermo Fisher Scientific, Waltham, MA, US), Taq DNA polymerase mix (20 mM Tris–HCl; pH 7.4 at 25°C, 0.1 mM EDTA, 1 mM DTT, 100 mM KCl, 200 μg/mL BSA, and 50% glycerol), 1X reaction buffer (1.5 mM MgCl2), 200 μM deoxynucleoside triphosphates (dNTPs), and 0.5 μM of each of the virulence gene primers. Five virulence genes (actA, hlyA, inlA, inlB, and plcB) were detected with PCR using the primers listed in Table 1 . PCR was performed on a Rotor-Gene Q thermal cycler (Qiagen) at the following conditions: initial denaturation at 98°C for 30 s, followed by 35 cycles of 98°C for 5 s, 60°C for 5 s, and 72°C for 10 s, with a final extension step at 72°C for 1 min. Amplified PCR products were loaded onto a 1.5% agarose gel and visualized under UV light.
4
Detection of Listeria virulence genes
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To detect virulence genes, such as inlA, inlB, actA, plcB, and hlyA, typical L. monocytogenes colonies on Palcam agar were resuspended in 100 μL of sterile distilled water and 50 μL of 0.05 N NaOH-0.25% sodium dodecyl sulfate solution and incubated at 99℃ for 15 min. For virulence gene amplification by PCR, an aliquot of this mixture (2 μL) was mixed with the components of the Phire Hot Start II DNA Polymerase Kit (Thermo Fisher), Taq DNA polymerase mix (20 mM Tris-HCl, pH 7.4 at 25℃; 0.1 mM EDTA; 1 mM DTT; 100 mM KCl; 200 μg/mL BSA; and 50% glycerol), 1× reaction buffer (1.5 mM MgCl2), 200 μM deoxynucleoside triphosphates (dNTPs; Promega Co., USA), and 0.5 μM each virulence gene primer listed in Table 1 . PCR was performed in a Rotor-Gene Q with the following program: an initial denaturation step at 98℃ for 30 s, followed by 35 cycles of 98℃ for 5 s, 60℃ for 5 s, and 72℃ for 10 s, with a final extension step at 72℃ for 1 min. After PCR, an aliquot of the reaction products (20 μL) was mixed with 4 μL of loading star, and then separated by electrophoresis in a 1.5% agarose gel.
5
Targeted Genomic Integration Validation
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Genomic DNA was extracted with the QIAamp DNA Blood Mini Kit (QIAGEN). G418 selected fibroblast and iPSC clones were analyzed for legitimate targeted integration by inside-out PCR using Phire Hot Start II DNA polymerase kit (Thermo Scientific). RNA was isolated with TRIzol (Life Technologies), and all RT-PCR reactions performed with the QuantiTect Reverse Transcription Kit (QIAGEN). All used primers are listed in S2 Table . For Southern blot analysis [62 (link)], genomic DNA was digested with EcoRV or BamHI, separated on a 0.8% agarose gel and transferred to Biodyne B nylon membrane (PALL Life Sciences). DNA was hybridized with a 32P-labeled fragment of PRE (for detection of the reprogramming vector) or NeoR (for detection of donor copies) using the DecaLabel DNA Labeling Kit (Fermentas). Labeled HindIII digested Lambda DNA was used as a marker.
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