Total RNA was isolated using the AxyPrep™ Purification Kit (Axygen, USA) according to the manufacturer’s instructions. The total RNA concentration and quality were measured with a Nanodrop 2000 micro-volume spectrophotometer (Thermo Scientific, USA) by absorbance measurements. RNA integrity was analyzed by 2% agarose gel electrophoresis and ethidium bromide staining. First-strand cDNA was synthesized from 3000 ng of total RNA using the RevertAidHMinus First Strand cDNA synthesis kit (Fermentas, USA) as instructed by the manufacturer. Real-time PCR (RT-PCR) reactions were carried out on an Mx3000P real-time PCR system (Stratagene USA). To create the RT-PCR standard, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the internal control. The PCR primer sequences were as follows: E2F1 primer sense 5′-CCC AAC TCC CTC TAC CCT-3′ and antisense 5′-CTC CCA TCT CAT ATC CAT CCT G-3′; and GAPDH primer sense 5′-ACC ACA GTC CAT GCC ATC AC-3′ and antisense 5′-TCA CCA CCC TGT TGC TGT A-3′. The PCR products were checked by agarose gel electrophoresis, and the abundance of each mRNA was detected and normalized to that of GAPDH mRNA.
Axyprep purification kit
Manufactured by Corning
Sourced in United States
The AxyPrep™ Purification Kit is a set of laboratory equipment designed for the purification of nucleic acids, such as DNA and RNA. The kit includes reagents and consumables necessary for the extraction and purification process. The core function of the kit is to enable efficient and reliable isolation of nucleic acids from various biological samples.
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Lab products found in correlation
3 protocols using axyprep purification kit
1
Quantitative Analysis of E2F1 Expression
2
RNA Isolation and RT-PCR Analysis
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For RNA isolation, the AxyPrep™ Purification Kit (Axygen, USA) was used according to the manufacturer's instructions. The concentration of total RNA was analysed by a Nanodrop 2000 micro-volume spectrophotometer (Thermo Scientific). Real-time PCR (RT-PCR) reactions were performed on an Mx3000P real-time PCR system (Stratagene USA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. The primers used to amplify the gene were: 5′-CAAAGTGACAGTGGGTGTGG-3′ and 5′-GCCAGGTCCTTCACTGTCTC-3′ for HK2; 5′-AC-CACAGTCCATGCCATCAC-3′ and 5′-TCACCACCCT-GTTGCTGTA-3′ for GAPDH.
3
Genomic DNA Methylation Analysis
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Genomic DNA was extracted from the lungs of the three deceased transgenic cloned kids and the normally produced kids using the TIANamp Genomic DNA Kit (Tiangen, Beijing, China), and this was followed by sodium bisulfite treatment using the Methylation-Gold™ Kit. Subsequently, the modified DNA sample was immediately used in bisulfite sequencing PCR (BSP) or stored at -80°C.
Specific primers, which were designed using MethPrimer, were used for the BSP amplification of IGF2R. Details of BSP-amplified nucleotide sequences of the IGF2R ICR are shown in Figure 1. PCR was performed with a DNA engine (Bio-Rad, USA) using the following program: 5 min at 95°C, followed by 40 cycles of denaturation for 30 s at 95°C, annealing for 30 s at 51°C, and extension for 30 s at 72°C. A final extension was run at 72°C for 5 min. The PCR products were gel-purified using the AxyPrep Purification Kit (Axygen, USA). The purified fragments were subcloned into pMD18-T vectors (TaKaRa, Japan). The clones confirmed by PCR were sequenced. We sequenced ten clones from each independent set of amplification and cloning. BSP results and C-T conversion rate were analyzed by the BIQAnalyzer software (http://biq-analyzer.bioinf.mpi-inf.mpg.de/). To ensure data quality, we chose sequences for which the C-T conversion rate was greater than 95% (Figure 1).
Specific primers, which were designed using MethPrimer, were used for the BSP amplification of IGF2R. Details of BSP-amplified nucleotide sequences of the IGF2R ICR are shown in Figure 1. PCR was performed with a DNA engine (Bio-Rad, USA) using the following program: 5 min at 95°C, followed by 40 cycles of denaturation for 30 s at 95°C, annealing for 30 s at 51°C, and extension for 30 s at 72°C. A final extension was run at 72°C for 5 min. The PCR products were gel-purified using the AxyPrep Purification Kit (Axygen, USA). The purified fragments were subcloned into pMD18-T vectors (TaKaRa, Japan). The clones confirmed by PCR were sequenced. We sequenced ten clones from each independent set of amplification and cloning. BSP results and C-T conversion rate were analyzed by the BIQAnalyzer software (http://biq-analyzer.bioinf.mpi-inf.mpg.de/). To ensure data quality, we chose sequences for which the C-T conversion rate was greater than 95% (Figure 1).
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