For bisulfite treatment, 1 μg genomic DNAs were denatured in 0.2 mol/L NaOH. Sodium bisulfite (Sigma) and hydroquinone (Sigma) were added to final concentrations of 3.1 mol/L and 0.5 mmol/L, respectively, and the samples were incubated at 55 °C for 16 h. After purification using DNA Clean-Up System (Promega), the DNA samples were desulfonated in 0.3 mol/L NaOH, precipitated with ethanol, and resolved with 20 μL Tris-EDTA (TE) buffer. The modified DNAs (20 ng) were amplified by PCR using Taq DNA polymerase (New England Biolabs, Beverly, MA). Cycling conditions were 94 °C for 5 min, followed by 35 cycles of 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 30 s, with a final extension of 5 min at 72 °C. The primer sequences for amplification of the IGF1 promoter region were 5′-GTTTAGAAGAGGATTTTTATGGGT-3’ (sense, upper strand) and 5′-CCTAACAAAAATATATCTTTAACTCC-3’ (antisense, upper strand). The resulting PCR products were cloned into a pGEM-T-easy vector (Promega) and subjected to sequencing analysis. The primer sequences for bisulfite sequencing, which encompasses the probe region used in pyrosequencing (+130 to +143 nt. from transcription start site), were 5’-GTGTTTTGTAGATAAATGTGAGGA-3’ (sense, upper strand) and 5’-CTACTAATTTTTCCCATTACTTCTA-3’ (antisense, upper strand).
Dna clean up system
Manufactured by Promega
Sourced in United States
The DNA Clean-Up System is a laboratory equipment designed to purify DNA samples. It effectively removes contaminants and impurities from DNA, preparing it for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Hydroquinone, by Merck Group (3 mentions)
Sodium bisulfite, by Merck Group (2 mentions)
Universal genomic dna extraction kit ver 3, by Takara Bio (2 mentions)
Pgem t easy vector, by Promega (1 mentions)
Taq dna polymerase, by New England Biolabs (1 mentions)
Pmd18 t vector, by Takara Bio (1 mentions)
Ex taq dna polymerase kit, by Takara Bio (1 mentions)
Pgfpuv, by Takara Bio (1 mentions)
Ptrc99a, by GE Healthcare (1 mentions)
6 protocols using dna clean up system
1
Bisulfite Sequencing of IGF1 Promoter
2
DNA Bisulfite Modification Protocol
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Methylation status of the CpG island was analyzed using a modified DNA bisulfite protocol [7 (link), 34 (link)]. DNA samples were denatured in
0.2 M NaOH at 37°C for 10 min. Sodium bisulfite was added to a final concentration of 3.0 M and hydroquinone
to a final concentration of 0.5 mM, followed by incubation at 55°C for 16 hr. DNA samples were then desalted
by the DNA Clean-Up System (Promega, Madison, WI, U.S.A.) followed by desulfonation in 0.3 M NaOH and
ethanol-precipitation.
For PCR, the initial denaturation was at 95°C for 4 min, followed by 40 cycles at 94°C for 30 sec, 53°C for
30 sec, 72°C for 30 sec, was followed by a final elongation at 72°C for 10 min. After PCR, the amplified DNA
fragments were separated by agarose gel electrophoresis and purified using gel recovery kit. The purified PCR
products were then ligated with pMD-18T vectors (Takara Co., Dalian, China). After amplification in
Escherichia coli, the plasmids were isolated and sequenced by Beijing Genome Institution.
Three clones from each sample were selected for sequencing.
0.2 M NaOH at 37°C for 10 min. Sodium bisulfite was added to a final concentration of 3.0 M and hydroquinone
to a final concentration of 0.5 mM, followed by incubation at 55°C for 16 hr. DNA samples were then desalted
by the DNA Clean-Up System (Promega, Madison, WI, U.S.A.) followed by desulfonation in 0.3 M NaOH and
ethanol-precipitation.
For PCR, the initial denaturation was at 95°C for 4 min, followed by 40 cycles at 94°C for 30 sec, 53°C for
30 sec, 72°C for 30 sec, was followed by a final elongation at 72°C for 10 min. After PCR, the amplified DNA
fragments were separated by agarose gel electrophoresis and purified using gel recovery kit. The purified PCR
products were then ligated with pMD-18T vectors (Takara Co., Dalian, China). After amplification in
Escherichia coli, the plasmids were isolated and sequenced by Beijing Genome Institution.
Three clones from each sample were selected for sequencing.
3
Genomic DNA Bisulfite Modification
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Genomic DNA from the cultured cells and specimens was isolated using a Universal Genomic DNA Extraction Kit Ver3.0 (Takara Bio Inc.). The concentration and purity of DNA were controlled by ultraviolet-visible spectrophotometry (1.82O and stored at −80°C. The genomic DNA of each specimen was treated with bisulfite modification and the protocol was as per Herman et al.25 (link) Genomic DNA (2 μg) was treated with sodium bisulfite (Sigma-Aldrich). In brief, genomic DNA was denatured in 3 mol/L NaOH for 15 minutes at 37°C. Cytosines were sulfonated in 3.6 mol/L sodium bisulfite and 1 mmol/L hydroquinone (Sigma-Aldrich) for 16 hours at 55°C. The modified DNA samples were desalted using a DNA cleanup system (Promega Corporation, Fitchburg, WI, USA). The modified DNA samples were dissolved with ddH2O and stored at −80°C.
4
Epigenetic Regulation of Sirt1 by MS-PCR
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Methylation-specific PCR (MS-PCR) was performed to evaluate the methylation status of Sirt1. Firstly, genomic DNA from the cultured cells was isolated using a Universal Genomic DNA Extraction Kit Ver3.0 (Takara Bio). The generated DNA of each sample was experienced bisulfite modification process as follows: degeneration in 3 mol/L NaOH for 15 min at 37°C; sulfonation of cytosines in 3.6 mol/L sodium bisulfite and 1 mmol/L hydroquinone (Sigma–Aldrich) for 16 h at 55°C. Following, modified DNA samples were desalted using a DNA clean-up system (Promega Corporation). After treatment, Sirt1 was amplified using Takara's Ex Taq™ DNA Polymerase kit basing on two primers of Sirt1 which cover almost the entire CpG rich region of the proximal Sirt1 promoter in Biomedical Instrumentation Center (USUHS). Relative level of MS-PCR products were analysed by a 2% agarose gel and normalized to non-methylation 16S RNA.
5
Bisulfite Treatment of DNA Protocol
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Bisulfite treatment of DNA was performed according to the reported method.43 (link) First, 2 μg of DNA was denatured in 0.35 M NaOH at 42 °C for 30 min. Bisulfite reaction was carried out in 3.2 M sodium bisulfite and 0.5 mM hydroquinone (both were freshly prepared) at 50 °C for 16–18 h. Then DNA was recovered with a desalting column (DNA cleanup system, Promega Inc., U.S.A.) and the modification was completed in 0.3 M NaOH at 37 °C for 15 min, followed by neutralization with ammonium acetate, precipitation with ethanol, and drying. The resulting DNA was resuspended in water and used immediately or stored at –20 °C.
6
Construction and Analysis of Metagenomic Library
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E. coli XL1-Blue (Stratagene, La Jolla, CA, USA) was used as the host for constructing the metagenomic DNA library and expressing foreign proteins. A previously constructed trap vector pBGRI, employed as a cloning vector for the construction of a soil metagenomic DNA library [13 ], was used for analyzing the expression patterns of deletion fragments of trapped DNA including expression modules (Figure 1 ). To amplify the corresponding DNA fragment containing the innately overexpressed ORF, the recombinant plasmid isolated from the screened clone was used as the template. A set of primers described in Table 1 and high fidelity Taq polymerase, Phusion (New England laboratory, Hitchin, UK) were used for PCR. The resulting DNA was purified by using a DNA clean-up system (Promega, Madison, WI, USA). pTrc99a (Pharmacia Biotech, Uppsala, Sweden) was used as the backbone plasmid to construct the pTB vector without any factors for gene expression. pQE30-1767 [14 (link)], pTrc99a-SmGlu [15 (link)], pMal-c2x Phusion (New England laboratory, Hitchin, UK), and pGFPuv (Clontech, Mountain view, CA, USA) were used as templates for PCR amplification of esterase 1767, β-glucosidase, maltose binding protein, and green fluorescence protein, respectively.
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