Total RNA was isolated from cultured RAW264.7 cells using TRIzol reagent (Ambion®, Carlsbad, CA, USA) according to the manufacturer’s instructions. First strand cDNA was synthesized from the total RNA (3 μg) using oligo dT (DiaStar RT Kit, SolGent, Daejeon, Korea) and was used as a template for PCR amplification with G-Taq polymerase (Cosmogenetech, Seoul, Korea). The first-strand cDNA was quantified using a spectrophotometer (NanoDrop® ND-1000, NanoDrop Technologies, Wilmington, DC, USA). The quantified cDNA was used as a template. PCR assay was performed with specific primers for inducible nitric oxide (iNOS), IL-1β, and TNF-α. The primer sequences are listed in Table S1 . The PCR steps included initial denaturation at 94 °C for 5 min, then 28 cycles at 94 °C for 30 s, 57 °C for 30 s, and 72 °C for 30 s, and a final extension step at 72 °C for 10 min. The amplified PCR products were separated in 1.5% agarose gel, and the gel was stained with ethidium bromide. The bands were visualized using the iBrightTM CL1500 imaging system (Thermo Scientific Fisher/Life Technologies Holdings Pte Ltd., Singapore). The DNA fragments were directly sequenced with the ABI PRISM® 3100-Avant Genetic Analyzer (Applied Biosystems, Carlsbad, CA, USA).
G taq polymerase
Manufactured by Cosmogenetech
G-Taq polymerase is a thermostable DNA polymerase used in polymerase chain reaction (PCR) experiments. It is a recombinant Taq DNA polymerase with enhanced fidelity and processivity. G-Taq polymerase can be used for a variety of PCR applications, including genotyping, DNA sequencing, and gene expression analysis.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Abi prism 3100 avant genetic analyzer, by Thermo Fisher Scientific (2 mentions)
Nanodrop nd 1000, by Thermo Fisher Scientific (2 mentions)
Diastar rt kit, by Solgent (2 mentions)
Riboex reagent, by GeneAll (2 mentions)
Improm iitm reverse transcriptase, by Promega (2 mentions)
Riboex, by GeneAll (2 mentions)
Superscript reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Sybr green master mix, by Takara Bio (1 mentions)
M mlv reverse transcriptase, by Cosmogenetech (1 mentions)
Riboex regent, by GeneAll (1 mentions)
Improm 2 reverse transcriptase, by Promega (1 mentions)
Improm iitm, by Promega (1 mentions)
Gel extraction kit, by Qiagen (1 mentions)
Ez dna methylation gold kit, by Zymo Research (1 mentions)
9 protocols using g taq polymerase
1
Analyzing Gene Expression in RAW264.7 Cells
2
Quantification of AIMP Genes in Mouse Sciatic Nerves
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Total RNA was isolated from mouse sciatic nerves using Tri Reagent (MRC, Cincinnati, OH, USA), and first-strand cDNA was obtained with SuperScript Reverse transcriptase (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s protocol. Real-time quantitative PCR (qPCR) was performed with SYBR green master mix (Takara, Kusatsu, Japan). Primers for qPCR are: AIMP1 (FW: 5’-TTTCTCTGCCGATTCTGGGGA-3’, RV: 3’-CCTGCTGCTTGAGATATTCGAT-5’), AIMP2 (FW: 5’-CGTGCAGGAAACATCCGA-3’, RV: 3’-GTTACGTCCAAGTCTGCATCT–5’), AIMP3 (FW: 5’-GACTGAAGCCGGGGAATAAGT-3’, RV: 3’-TAGACTCGGGCCATTGTTTGT–5’) and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (FW: 5’–TGCACCACCAACTGCTTAGC-3’, 3’-GGCATGGACTGTGGTCATGA–5’). Semi-PCR was performed using G-Taq polymerase (Cosmogenetech, Seoul, Korea). Primers for semi-PCR are: AIMP1 (FW: 5’-AAAAGGAGAAGCAGCAGTCG-3’, RV: 5’-TCTGGTGAACTGGCACACAT-3’), AIMP2 (FW: 5’-AGTTGAAGGCAGCAGTCGAT-3’, RV: 5’-GACAAGATTCTCGGGCACAT-3’), AIMP3 (FW: 5’-ATCGCCACCCATCTAGTCAA-3’, RV: 5’-GACAAAACCAGCGAGACACA-3’), GAPDH (FW: 5’-CTACATGGTCTACATGTTCCAGTATG-3’, RV: 5’-AGTTGTCATGGATGACCTTGG-3’).
3
RNA Extraction and Splicing Analysis
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Total cellular RNA was extracted using the RiboEx reagent (GeneAll) according to the manufacturer’s instructions. 1 μg of total RNA was reverse transcribed using M-MLV reverse transcriptase (ELPiS) and amplified by PCR using G-Taq polymerase (Cosmo Genetech). The following primers were used for splicing studies: Exon6.F (5′-ATAATTCCCCCACC ACCTCC-3′), Exon8.R (5′-ACTACAACACCCTTCTCACAG-3′), pcDNA.F (5′-CACTGCTTACTGGCTTATCGAA-3′), pcDNA.R (5′-CTAGAAGGCACAGTCGAGGCT-3′), AdML.F (5′-ACTCTT GGATCGGAAACCCGT-3′).
4
Analyzing Splicing Patterns in Cell Lines
5
RNA Extraction and cDNA Synthesis
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Total RNA was extracted using RiboEx (GeneAll) as previously described (23 (link)). Reverse transcription was performed using 0.5 μg RNA with oligo (dT) primer and ImProm-IITM reverse transcriptase (Promega). The reaction mixture (0.5 μl) was amplified by PCR using G-Taq polymerase (Cosmo Genetech).
6
Quantification of SMN Minigene Splicing
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RiboEx reagent (GeneAll) was used to extract total RNA transfected cells. 1 μg of RNA sample was used per 2 μL reaction with oligo (dT) primer and ImProm-IITM reverse transcriptase (Promega). 1 μL of cDNA was amplified by PCR with G-Taq polymerase (Cosmo Genetech). The primers for SMN1-S, SMN2-S minigene, and conserved splice site constructs are as follows: forward: SMN.Fwd (5′-ATA ATT CCC CCA CCA CCT CC-3′), reverse: pCDNA.Rev (5′-CTA GAA GGC ACA GTC GAG GCT-3′) which specifically anneal to the plasmid backbone. The GAPDH mRNA was amplified with primers GAPDHFwd (5′-ACC ACA GTC CAT GCC ATC A-3′) and GAPDHRev (5′-TCC ACC ACC CTG TTG CTG TA-3′). Splicing products were amplified using 20 PCR cycles (94°C for 30 s, 60°C for 30s, and 72°C for 30 s). The PCR products from SMN minigene constructs were loaded onto 2% agarose gels. These gels could be visualized by staining with ethidium bromide solution (0.5 μg/mL). Intensity of the approximate bands was quantified using the ImageJ Software (National Institutes of Health, Bethesda, MD).
7
Bisulfite Sequencing of BORIS Promoter
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Genomic DNA was subjected to bisulfite modification using the EZ DNA Methylation-Gold Kit (Zymo Research, Irrine, CA, USA). Each sequence was amplified using G-Taq polymerase (Cosmo Genetech, Seoul, Korea) as previously described.6 (link) PCR fragments were gel extracted using a Gel Extraction kit (Qiagen) and ligated into the Mighty TA cloning vector (TaKaRa, Kusatsu, Shiga, Japan). Following transformation, plasmids from individual bacterial colonies were isolated and sequenced. Using bisulfate sequencing, the methylation status of the BORIS promoter region was determined and represented as follows: open circles indicate non-methylated CpG and filled circles indicate methylated CpG (Figure 1 ).
8
RNA Extraction and RT-PCR Analysis
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Total RNA was extracted using RiboEx (GeneAll) following the manufacturer's protocol. Reverse transcription was performed using 0.5 ㎍ RNA with oligo (dT) primer and ImProm-IITM reverse transcriptase (Promega). 0.5µl of the reaction mixture was amplified by PCR using G-Taq polymerase (Cosmo Genetech). Spliced products of minigenes were detected with Exon4.F and pcDNA.R primers. All of primer sequences are listed in Table 1 .
9
Transcriptional Profiling of Macrophage Polarization
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Total RNA from the cells was extracted with Trizol reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. First strand cDNA was synthesized from the total RNA (3 μg) isolated from the RAW264.7 cells, the mouse peritoneal and human THP-1-differentiated macrophages, and the TAMs using oligo dT (DiaStar RT Kit, SolGent, Daejeon, Korea). The first-strand cDNA was quantified using a spectrophotometer (NanoDrop® ND-1000, NanoDrop Technologies, Wilmington, DE, USA). The quantified cDNA was used as a template for PCR amplification with G-Taq polymerase (Cosmogenetech, Seoul, Korea). PCR assay was performed with specific primers for M1 and M2 polarization markers. The specific primer sequences are listed in Table 1 . The PCR steps included initial denaturation at 94 °C for 5 min, then 32 cycles at 94 °C for 30 s, 55 °C to 60 °C (see Table 1 ) for 30 sec, 72 °C for 30 sec, and a final extension step at 72 °C for 10 min. The amplified PCR products were separated in 1.5% agarose gel stained with ethidium bromide. The bands obtained by RT-PCR were extracted and directly sequenced with an ABI PRISM® 3100-Avant Genetic Analyzer (Applied Biosystems, Carlsbad, CA, USA).
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