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7 protocols using hpaii

1

Amplified Fragment Length Polymorphism and Methylation-Sensitive Amplification Polymorphism Protocols

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The AFLP digestion reaction comprised 3 U PstI and 3 U MseI (Li-COR, Co., Lincoln, NE, USA), the pre-amplification and selective amplification reaction conditions and the method of electrophoresis were adapted from Cao et al. [25] . The AFLP adapter and selective amplification primer sequences are listed in Table S1.
The MSAP experiment comprised two digestion reactions, the first digestion reaction included 16 U of EcoRI (TaKaRa, Dalian, China) plus 10 U of MspI (TaKaRa), the second digestion reaction was the same as the first digestion except the HpaII (TaKaRa) instead of MspI. The MSAP pre-amplification and selective amplification reaction conditions and the method of electrophoresis were followed by Cao et al. [26] . The MSAP adapter and selective amplification primer sequences are listed in Table S2.
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2

X-Chromosome Inactivation Pattern Analysis

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X chromosome inactivation (XCI) pattern analysis was used to isolate CpG dinucleotides with cytosine methylation that are located within the polymorphic CAG repeat of the human androgen receptor (AR) gene promoter region. The MIC promoter region (375 bp) was used to establish complete digestion, and the proband's DNA sample was included as a control. The DNA was digested with the methylation‐sensitive restriction enzyme HpaII (Takara, Japan). The PCR primers included: AR‐FP: 6‐FAM‐TCCAGAATCTGTTCCAGAGCGTGC, AR‐RP: GCTGTGAAGGTTGCTGTTCCTCAT, MIC2‐FP: 6‐FAM‐AGAGGTGCGTCC GATTTTTCCC, and MIC2‐RP: ACCGCCGCAGATGGACAATT. The amplicons were then sequenced using an ABI 3730 (Applied Biosystems Inc.) and analyzed using GeneMarker software (SoftGenetics). XCI status was determined by performing peak comparisons between the two AR alleles (different sizes) from digested and nondigested DNA aliquots for each sample (Jones, 2014).
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3

Histochemical Staining with DAB and DAPI

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Paraformaldehyde (PFA) was purchased from Merck (Darmstadt, Germany), and 3,3'-diaminobenzidine 4HCl (DAB) was purchased from Dojin Chemical Co. (Kumamoto, Japan). Bovine serum albumin (BSA) (essentially fatty acid and globulin-free), Trizma base and Brij-35 were from Sigma Chemical Co. (St. Louis, MO, USA). Biotin-16-dUTP, digoxigenin-11-dUTP and terminal deoxynucleotidyl transferase (TdT) were from Roche Diagnostics (Mannheim, Germany). Dideoxy ATP (ddATP) and dideoxy TTP (ddTTP) were from Jena Bioscience (Jena, Germany). Hpa II, Msp I, Sau3A I and Mbo I were purchased from Takara Bio Inc. (Shiga, Japan). 4',6-Diamidino-2-phenylindole (DAPI) was from DAKO (Glostrup, Denmark). Permount was purchased from Fisher Scientific Inc. (NJ, USA). All other reagents used in this study were from Wako Pure Chemicals (Osaka, Japan) and were of analytical grade.
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4

Methylation Analysis of AR Gene

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DNA was extracted from female patients with DDCS according to the above procedure, and normal female blood sample DNA was used as a control. The digestion reaction system (final volume = 20 μl) included 10 μl DNA, 1 μl HpaII (Takara, Dalian, China), 2 μl 10× loading buffer, and 7 μl dH2O. Distilled water instead of enzyme was used in the nondigested system as a control. The reaction conditions were 37 °C digestion for 3 hours and then incubation at 85 °C for 15 minutes to stop the reaction. This was followed by PCR using a 50 μl reaction system including 10 μl 5× PrimeSTAR GXL Buffer, 4 μl dNTP mix, 1 μl PrimeSTAR GXL DNA polymerase (Takara, Dalian, China), 1 μl each primer (AR) (Sangon Biotech, Shanghai, China), 28 µl sterile water, and 5 μl digested or nondigested DNA. The sequences of the primers were 5′-CTACCGAGGAGCTTTCCAGAAT-3′ (forward primer, labeled with FAM green fluorescence on the 5′ end) and 5′-CGATGGGCTTGGGGAGAACCAT-3′ (reverse primer). The mixture underwent predenaturation at 95 °C for 5 minutes, followed by 30 cycles at 98 °C for 10 s and 68 °C for 30 seconds. The PCR product was used for capillary electrophoresis on an ABI 3730 sequencer (Applied Biosystems), and the data were analyzed by GeneMapper 4.0 Software (Applied Biosystems).
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5

Genomic DNA Extraction and Methylation Analysis

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We obtained the genomic DNA of the brain cortex using a DNA extraction kit (KG203, TIANGEN BIOTECH Co., Ltd., Shanghai, China). The target fragment was subjected to restriction analysis using DNAMAN software (Lynnon Biosoft, San Ramon, CA, USA), and the genomic DNA was pre-cut by restriction enzymes that did not cleave the target fragment. This study used EcoRI (cat. 1040S, Takara, Dalian, Liaoning Province, China) to pre-cut the genomic DNA. We used MspI and HpaII (cat. 1150A, Takara, Dalian, Liaoning Province, China) to process the mass of each sample equalized after digestion, and each sample was made with three parallels, MspI, HpaII, and a blank control tube. Quantification of the absolute copy number of the genomic DNA was performed after enzyme treatment with RT-PCR and the calculation of the methylation of sample’s promoter. The primers used for methylation detection are listed in Supplemental Table S3.
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6

Immunochemical Detection of DNA Modifications

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Bovine serum albumin (BSA; essentially fatty acid and globulin-free), Trizma base, 2-mercaptoethanol, 3-aminopropyltriethoxysilane and Brij-35 were obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany); 3,3′-Diaminobenzidine-4HCl (DAB) was purchased from Chemical Dojin Co., Ltd. (Kumamoto, Japan). Biotin-16-dUTP, digoxigenin-11-dUTP, Rhodamine anti-Dig and terminal deoxynucleotidyltransferase (TdT) were from Roche Diagnostics (Mannheim, Germany). Dideoxy ATP (ddATP) and dideoxy TTP (ddTTP) were from Jena Bioscience (Jena, Germany). HpaII and MspI were purchased from Takara Bio, Inc. (Otsu, Japan). DAPI was obtained from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Permount was from Thermo Fisher Scientific, Inc. All other reagents used in the present study were obtained from Wako Pure Chemicals Industries, Inc. (Osaka, Japan) and were of analytical grade.
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7

FRAXA Locus Methylation Assay for XCI Analysis

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We performed XCI analysis using FRAXA locus methylation assay as previously described (62 (link)). Briefly, genomic DNA from the subjects was digested with two methylation-sensitive enzymes, Hpa II and Hha I (Takara). The digested DNA was PCR-amplified with fluorescent-labeled primers targeting a triplet repeat of FRAXA gene. See table S6 for a list of primer sets. Fluorescent-labeled products were analyzed on an ABI PRISM 3500 Genetic Analyzer with GeneMapper Software version 4.0 (Applied Biosystems). X-inactivation ratios of less than or equal to 80:20 were considered to represent a random pattern, ratios greater than 80:20 were considered to represent a skewed pattern, and ratios greater than 90:10 were considered to represent a markedly skewed pattern. Fragment length along with an appropriate standard was determined using a 3730xl DNA Analyzer (Applied Biosystems) and analyzed with Peak Scanner Software 2 (Applied Biosystems).
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