ChIP assays for histone methylation and HP1a recruitment were performed using an EZ-Magna ChIP G Kit (Millipore, CA). Briefly, monoclonal Hep3B cells with stable expression of copGFP in 10-cm dishes were transiently transfected with 15 μg suppressor vectors. At 48 hr after transfection, cells were cross-linked with 1% formaldehyde (Sigma, MO) and harvested for immunoprecipitation. Antibodies used in ChIP assays included anti-H4K4Me3, anti-H3K9Me3, anti-H3K27Me3, and anti-HP1a (Millipore, CA).32 (link) An aliquot of cell lysates was saved to serve as the input DNA control. After the reversal of cross-linking at 62°C for 2 hr and 95°C for 10 min, ChIP samples were purified and subjected to real-time qPCR. Individual ChIP assays were repeated three times to confirm the reproducibility of the qPCR. Real-time qPCR was performed using 2xKapa mixed with SYBR (Applied Biosystems, CA) on an ABI PRISM 7900 HT Sequence Detection System (Applied Biosystems, CA) with GRN primers (forward, 5′-GCGGTTTTGGCAGTACATCA-3′; reverse, 5′-GGGCGGAGTTGTTACGACAT-3′). Individual ChIP assays were repeated three times to confirm the reproducibility of the qPCR.
2xkapa
Manufactured by Thermo Fisher Scientific
Sourced in United States
2xKapa is a premixed, ready-to-use 2X concentrated PCR master mix that contains all the necessary reagents for DNA amplification, except for the template DNA and primers. The master mix includes a high-fidelity DNA polymerase, dNTPs, and buffer components optimized for efficient and reliable PCR.
Automatically generated - may contain errors
Lab products found in correlation
Abi prism 7900ht sequence detection system, by Thermo Fisher Scientific (3 mentions)
Anti h3k9me3, by Merck Group (2 mentions)
Anti h4k4me3, by Merck Group (2 mentions)
Anti hp1a, by Merck Group (2 mentions)
M mlv reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Ez magna chip g kit, by Merck Group (1 mentions)
Anti h3k27me3, by Merck Group (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Dneasy blood tissue kit, by Qiagen (1 mentions)
Ez dna methylation gold kit, by Zymo Research (1 mentions)
Ez magna chip g chromatin immunoprecipitation kit, by Merck Group (1 mentions)
Anti h3k27me2, by Merck Group (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
3 protocols using 2xkapa
1
Chromatin Immunoprecipitation for Histone Modifications
2
Quantitative Gene Expression Analysis
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Genomic, bisulfate-converted DNA and mRNA were extracted using DNeasy Blood & Tissue Kit (QIAGEN, CA), EZ DNA MethylationGold kit (Zymo Research, CA), and RNeasy Mini Kit (QIAGEN, CA), and mRNAs were reverse-transcribed using M-MLV Reverse Transcriptase (Thermo Fisher Scientific, CA). Real-time qPCR was performed using 2xKapa mixed with SYBR (Applied Biosystems, CA) on an ABI PRISM 7900 HT Sequence Detection System (Applied Biosystems, CA) with primers. All experiments were performed in triplicate. PCR primers used for qPCR are listed in Table S1 .
3
ChIP Assay for Histone Modifications
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As described previously [21 (link),51 (link)], ChIP assays for histone methylation and HP1a recruitment were performed using an EZ-Magna ChIP™ G chromatin immunoprecipitation Kit (Millipore, California, United States). Briefly, monoclonal 293 T cells for stable expression of copGFP in 10 cm dishes were transiently transfected with 15 μg of various suppressor vectors. Forty eight hours after transfection, cells were cross-linked with 1% formaldehyde (Sigma, Missouri, United States) and harvested for immunoprecipitation. Antibodies used in ChIP assays included anti-H4K4Me3, anti-H3K9Me3, anti-H3K27Me2, and anti-HP1a (Millipore, California, United States). An aliquot of cell lysates was saved to serve as the input DNA control. After the reversal of crosslinking at 62°C for 2 hours and 95°C for 10 minutes, ChIP samples were purified and subjected to real-time qPCR. Individual ChIP assays were repeated three times to confirm the reproducibility of the qPCR. Real-time qPCR was performed using 2xKapa mixed with SYBR (Applied Biosystems, California, United States) on an ABI PRISM 7900 HT Sequence Detection System (Applied Biosystems, California, United States) with pCMV primers (forward, 5’-gcggttttggcagtacatca-3’; reverse, 5’-gggcggagttgttacgacat-3’). Each sample was analyzed in quadruplicate.
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