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Found 4 matching solutions for this experiment
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For 2D agarose gel analysis, 10μg DNA was digested overnight at 37°C with 10U HinfI (Thermo Fisher), 10U RsaI (Thermo Fisher) and 2μg/mL RNase A (Takara). The reaction was terminated with EDTA and analyzed by 2D agarose gel electrophoresis. 30U RecJf (New England Biolabs) was added for removing 5' single-stranded DNA.
For internal gaps/nicks analysis, 5μg genomic DNA was digested overnight at 37°C with 5U HinfI (Thermo Fisher), 5U RsaI (Thermo Fisher) and 1μg/mL Ribonuclease A (RNase A, Takara) and purified with QIAquick PCR Purification kit (Qiagen). Purified DNA were digested with or without 200U Exonuclease III (New England Biolabs) overnight at 37°C, and then subjected to 0.7% agarose gel electrophoresis and in-gel hybridization. |
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Protocol tips |
For 2D agarose gel analysis, 10μg DNA was digested overnight at 37°C with 10U HinfI (Thermo Fisher), 10U RsaI (Thermo Fisher) and 2μg/mL RNase A (Takara). The reaction was terminated with EDTA and analyzed by 2D agarose gel electrophoresis. 30U RecJf (New England Biolabs) was added for removing 5' single-stranded DNA.
For internal gaps/nicks analysis, 5μg genomic DNA was digested overnight at 37°C with 5U HinfI (Thermo Fisher), 5U RsaI (Thermo Fisher) and 1μg/mL Ribonuclease A (RNase A, Takara) and purified with QIAquick PCR Purification kit (Qiagen). Purified DNA were digested with or without 200U Exonuclease III (New England Biolabs) overnight at 37°C, and then subjected to 0.7% agarose gel electrophoresis and in-gel hybridization. |
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According to the SNP mutation site gene, we selected the restriction enzyme HinfI (Takara Bio, Inc.) for the treatment of PCR amplified fragments, to carry on the identification of the gene. All of the 180 GDM and 210 in the control group PCR amplified fragments were by HinfI. The reaction system contained Takara HinfI 1 μl, 10X HinfI buffer 2 μl, PCR template 7-8 μl, add ultrapure water to 20 μl, kept in 37°C water for 8 h. The fragments digested by HinfI were electrophoresed in a 3% agarose gel. Genotypes were determined by the results of various bands though UV light (c150; Azure Biosystems, Inc.). |
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Protocol tips |
According to the SNP mutation site gene, we selected the restriction enzyme HinfI (Takara Bio, Inc.) for the treatment of PCR amplified fragments, to carry on the identification of the gene. All of the 180 GDM and 210 in the control group PCR amplified fragments were by HinfI. The reaction system contained Takara HinfI 1 μl, 10X HinfI buffer 2 μl, PCR template 7-8 μl, add ultrapure water to 20 μl, kept in 37°C water for 8 h. The fragments digested by HinfI were electrophoresed in a 3% agarose gel. Genotypes were determined by the results of various bands though UV light (c150; Azure Biosystems, Inc.). |
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RFLP was carried out in 30 μl reaction containing 0.5 μg (10-15 μl) PCR product, RE buffer, 2U of HaeIII / HinfI / XhoI restriction enzyme from NEB [9, 12]. Digestion was carried out for 1 h 30 min. Digests were electrophoresed on 1% agarose gel and band patterns were analyzed by visual comparison. |
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RFLP was carried out in 30 μl reaction containing 0.5 μg (10-15 μl) PCR product, RE buffer, 2U of HaeIII / HinfI / XhoI restriction enzyme from NEB [9, 12]. Digestion was carried out for 1 h 30 min. Digests were electrophoresed on 1% agarose gel and band patterns were analyzed by visual comparison. |
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Aliquots of 10 μL of each 275 bp amplified product were digested with restriction endonucleases as recommended by the manufacturer. Three restriction enzymes, DdeI, HaeIII and HinfI (Promega, Madison, WI, USA), were used in separate reactions. |
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Aliquots of 10 μL of each 275 bp amplified product were digested with restriction endonucleases as recommended by the manufacturer. Three restriction enzymes, DdeI, HaeIII and HinfI (Promega, Madison, WI, USA), were used in separate reactions. |
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