Total RNAs from MEFs, iPS cells and ES cells were prepared by using RNApure Rapid Extraction Kit (BioTeke Corporation, Beijing, China) according to the instructions of the manufacturer. Reverse transcription of equal total RNA was performed using oligo (dT) primer and Super M-MLV reverse transcriptase (BioTeke Corporation) to generate complementary DNA (cDNA). Then, cDNA was amplified by using reverse transcription- polymerase chain reaction (RT-PCR) with the specific primers and Taq PCR Master-mix (BioTeke Corporation), which was performed in Life Express PCR (BIOER, Hangzhou, China). The primer information used is presented in Table 1 . β-actin was used as the internal control gene. The reverse transcription- PCR conditions were as follows: (1) an initial incubation at 95°C for 5 min, (2) denaturation at 95°C for 20 s, (3) annealing at 54°C for 20 s, and (4) extension at 72°C for 30 s. Steps (2–4) were repeated for 35 cycles, followed by a final polymerization at 25°C for 5 min. Subsequently, the PCR amplification products (Oct4, Sox2, Nanog, Klf4, Fbx15, and β-actin) were visualized in 1.5% agarose gel and analyzed using Gel Imaging Analyzer (Liuyi, Beijing, China).
Taq pcr master mix
Manufactured by BioTeke
Sourced in China
Taq PCR Master-mix is a ready-to-use solution containing all the necessary components for performing Polymerase Chain Reaction (PCR) amplification, including Taq DNA polymerase, dNTPs, and buffer. It is designed to simplify the PCR setup process and ensure consistent results.
Automatically generated - may contain errors
4 protocols using taq pcr master mix
1
Characterization of Pluripotency Markers in MEFs, iPS, and ES Cells
2
SNP Genotyping by PCR-RFLP
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First, 200 μL of EDTA-anticoagulated peripheral blood samples from each participant was used to extract the genomic DNA using a DNA isolation kit (BioTeke, Peking, China) following the manufacturer's protocol. The genotyping of the two selected SNPs (rs12934561 and rs28372698) was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Primers were designed with Primer 3 web version 4.1.0. (http://primer3.ut.ee/ ) [24 ] (Table 2 ). DNA fragments with polymorphisms were amplified in a 10 μL volume reaction system with 100 ng of extracted genomic DNA, 2.7 pico mole of forward and reverse primers of each SNP, and 5 μL 2x power Taq PCR Master Mix (BioTeke, Peking, China). The PCR annealing temperature of the two SNPs was set at 60°C for 30 s. After the PCR, products were digested by restriction enzyme Hpy188III at 37°C for 4 h. Finally, the digested fragments were separated by a 6% polyacrylamide gel and stained with 1.5 g/L argent nitrate [25 (link)]. To confirm the genotypes, we performed the DNA sequencing analysis, and approximately 10% of randomly selected samples were 100% in agreement with the results.
3
Quantitative RT-PCR Analysis of GIMAP Genes
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cDNA templates of different genes were obtained by RT of the RNA using Super M-MLV RT (BioTeke Corporation, Beijing, China), and the final reaction mixture of volume 20 µl contained 10 µl of 2X Taq PCR Master-mix (BioTeke Corporation), 1 µl of each primer (GIMAP5, forward 5′-CATGTTAGGGAAGCTCAGTC-3′, reverse 5′-GAAGGGTTCTACTGTGTCTCA-3′; GIMAP6, forward 5′-TGGATGCTCTGGATGTTGCA-3′, reverse 5′-TCCTGCTCATCCCCTTGTG-3′; hypoxanthine phosphoribosyltransferase 1 (HPRT1), forward 5′-GACCAGTCAACAGGGGACAT-3′, reverse 5′-GTGTCAATTATATCTTCCACAATCAAG-3′), 1 µl cDNA template and 7 µl of RNase free H2O. Thermal cycling parameters for the amplification were as follows: Denaturation step, 95°C for 5 min; followed by 36 cycles at 95°C for 20 sec, 52°C for 20 sec and 72°C for 30 sec; and the reaction was stopped by a step at 25°C for 5 min. The relative expression level of the targeted genes was normalized to HPRT1 according to the 2−ΔΔCq method (13 (link)).
4
FOXP3 ChIP-PCR Assay Protocol
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A ChIP assay was performed with a Cell ChIP kit (Wanleibio Co., Ltd.) according to the manufacturer's instructions. Briefly, MDA-MB-231 cells were fixed in 1% paraformaldehyde at room temperature for 10 min and sonicated. Next, the cell lysates were incubated with anti-FOXP3 antibody (undiluted, cat. no. PA1-806; Thermo Fisher Scientific, Inc.) or negative control IgG (undiluted) at 4°C overnight, then mixed with 60 µl protein A beads at 4°C. The immunoprecipitated complex was washed from beads through centrifuging at 625 × g for 1 min at 4°C and the supernatant was collected. The target DNA fragments were then amplified by polymerase chain reaction (PCR), analyzed by 2% agarose gel electrophoresis and visualized using Gold View nuclear staining dye. The PCR reaction system contained 2 µl immunoprecipitated DNAs, 1 µl forward primers, 1 µl reverse primers, and 10 µl 2X Taq PCR Master-mix (BioTeke Corporation), as well as sterile ultra-pure water to adjust total volume to 20 µl. The primer sequences were listed in Table SII . The PCR amplifications were performed under the following conditions: 95°C for 5 min; 40 cycles of 20 sec at 95°C, 20 sec at 50°C, 30 sec at 72°C; 25°C for 5 min.
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