96 well block module

Manufactured by Thermo Fisher Scientific

Sourced in United States

The 96-well block module is a standard laboratory equipment used for various sample processing and analysis applications. It provides a platform for holding and incubating 96 individual samples or reactions simultaneously in a microplate format.

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7900HT Fast Real-Time PCR System with Fast 96-Well Block Module Fast 96-Well Block Module 96-block module

7 protocols using 96 well block module

Chromatin Immunoprecipitation (ChIP) Assay

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A chromatin immunoprecipitation (ChIP) assay was carried out using the ChiP-IT Express Enzymatic Magnetic Chromatin Immunoprecipitation Kit or the Re-ChIP-IT kit (Active Motif, Carlsbad, CA) according to the manufacturer’s protocol. Approximately 300 mg of liver tissue was minced, homogenized, and fixed with 1% formaldehyde for the in vivo ChIP assay. The following ChIP primer sets were used for the ChIP assay: human AHR ChIP primer set I (−283 to 90, forward): 5′-TTA GCT GAC CCA CCG TCT CT-3′, (−283 to −90, reverse): 5′-TCC ATT CCG TCT TCC TTG AG-3′; human AHR ChIP primer set II (−1969 to −1779, forward): 5′-TTA GCT GAC CCA CCG TCT CT-3′, (−1969 to −1779, reverse): 5′-TTG GCT ATT TGG TGC AGT CA-3′; Mouse AHR ChIP primer set (−138 to +141, forward): 5′-AGA ACC TCG GAC TGC AAG AA-3′, (−138∼ +141, reverse): 5′-AGT CCG TCC ACC AGT TCG T-3′. For ChIP assays, NR2E3 antibody from Santa Cruz Biotech (Cat#: sc-292264) or Aviva Systems Biology (Cat#: ARP39069) was employed. All the ChIP-PCR reactions were performed using a 7300HT Real-Time PCR System with a 96-well block module (Applied Biosystems). The cycling conditions were 56 °C for 30 min and 95 °C for 10 min, followed by 50 cycles of 95 °C for 25 s and 60 °C for 60 s.

Khanal T., Choi K., Leung Y.K., Wang J., Kim D., Janakiram V., Cho S.G., Puga A., Ho S.M, & Kim K. (2017). Loss of NR2E3 represses AHR by LSD1 reprogramming, is associated with poor prognosis in liver cancer. Scientific Reports, 7, 10662.

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Chromatin Immunoprecipitation of Estrogen Receptors

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The livers were quickly removed, and 300 mg of liver was minced. A chromatin immunoprecipitation (ChIP) assay was then carried out using the ChiP-IT Express Enzymatic Magnetic Chromatin Immunoprecipitation Kit (Active Motif, Carlsbad, CA) according to the manufacturer’s protocol. The following Chip primer sets were used for the Chip assay: hESR1 II (forward): 5′-GCT GGA GCC CCT GAA CCG TCC GC-3′, hESR1 II (reverse): 5′-GGC CCA GAC TCC GAC GCC GCA-3′ (Park et al, 2012 (link)); mESR1 II (forward): 5′-CCT CCC GCC TTC TAC AGG T-3′, mESR1 II (reverse): 5′-CAC ACG GCA CAG TAG CGA G-3′. Chip-PCR was performed using the 7300HT Real-Time PCR System with a 96-well block module (Applied Biosystems) and Primer II. The cycling conditions were 56°C for 30 min and 95°C for 10 min, followed by 50 cycles of 95°C for 25 s and 60°C for 60 s.

Khnal T., Kim D., Johnson A., Choubey D, & Kim K. (2015). Deregulation of NR2E3, an orphan nuclear receptor, by benzo(a)pyrene-induced oxidative stress is associated with histone modification status change of the estrogen receptor gene promoter. Toxicology letters, 237(3), 228-236.

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Quantifying Cell Proliferation Markers

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Total RNA from cultured cells was extracted using a NucleoSpin RNA kit (740709; Macherey Nagel, Düren, Germany). Reverse transcription was performed on 500 ng of total RNA using a High Capacity cDNA Reverse Transcription Kit (4368813, Applied Biosystems). Real-time qPCR was achieved with the 7900HT Fast Real-Time PCR System with 96 Well Block Module (Applied Biosystems). The mRNA expression of 3 target genes was analyzed using predesigned and optimized inventoried assays (Applied Biosystems) according to the manufacturer’s instructions. The following assays were used: (Hs00606991_m1) Ki67 marker of proliferation; (Hs00923894_m1) P16 and (Hs01121172_m1) P21 markers of cyclin dependent kinase inhibitor; and to normalize gene expression, β-2-microglobulin (B2M) (Hs99999907_m1) was used as a housekeeping gene.

Lotti R., Palazzo E., Quadri M., Dumas M., Schnebert S., Biondini D., Bianchini M.A., Nizard C., Pincelli C, & Marconi A. (2022). Isolation of an “Early” Transit Amplifying Keratinocyte Population in Human Epidermis: A Role for the Low Affinity Neurotrophin Receptor CD271. Stem Cells, 40(12), 1149-1161.

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MALAT1 Promoter ChIP Assay with AHR and RNAPII

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A chromatin immunoprecipitation (ChIP) assay was carried out with AHR (Enzo Life Sciences, Inc., Cat#: ALX-804-423-R100) and RNA polymerase II (Active Motif, Carlsbad, CA, Cat# 39097) antibodies using Panc-1 cell ChIP lysate treated with TCDD for 2 hour with the ChIP-IT Express Chromatin Immunoprecipitation Kit (Active Motif), according to the manufacturer’s protocol. The ChIP primer set that covers MALAT1 gene promoter proximal region (−442~−201): (forward): 5’-AGGAGAGAGGTGGGAAAGGAAG-3’ and (reverse) 5’-TGGTTCTAACCGGCTCTAGC-3’. All the ChIP-PCR reactions were carried out using a 7300HT Real-Time PCR system or a QuantStudio 3 Real Time PCR system with a 96-well block module (Applied Biosystems). The cycling conditions were 56°C for 30 min and 95°C for 10 min, followed by 48 cycles of 95°C for 25 s and 60°C for 60 s.

Lee J.E., Cho S.G., Ko S.G., Ahrmad S.A., Puga A, & Kim K. (2020). Regulation of a long noncoding RNA MALAT1 by Aryl Hydrocarbon Receptor in pancreatic cancer cells and tissues.. Biochemical and biophysical research communications, 532(4), 563-569.

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Chromatin Immunoprecipitation Assay Protocol

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The chromatin immunoprecipitation (ChIP) assay was carried out using ChiP-IT Express Magnetic Chromatin Immunoprecipitation Kit (Active Motif, Carlsbad, CA) according to manufacturer's protocol. The Chip primer sets used for Chip assay: Primer I (forward: − 1558), 5′-TTG CCA CAT GAA GCA ATA GG-3′, Primer I (reverse: − 1090), 5′-CAG ACC CTT CCA CTG TTC GT-3′; Primer II (forward: − 458), 5′-CTG GTT CAA ACT TGG CTT CC-3′, Primer II (reverse: − 214), 5′-CTC CAC TGC CTT CTG AGT CC-3′; Primer III (forward: + 607), 5′-AGT CCG CAC TCA GAC AAA GG-3′, Primer III (reverse: + 1189) 5′-CAA GTT GGC CAA AAC AGC TT-3′. The positive (GAPDH) and negative primer (CNAP1) sequences were previously described [10] (link), [11] (link). Chip-PCR was performed using the 7300HT Real-Time PCR System with a 96-well block module (Applied Biosystems) with Primer II. Cycling conditions were 56°C for 30 min and 95°C for 10 min, followed by 50 cycles of 95°C for 25 s and 60°C for 60 s.

Yang W.S., Chadalapaka G., Cho S.G., Lee S.O., Jin U.H., Jutooru I., Choi K., Leung Y.K., Ho S.M., Safe S, & Kim K. (2014). The Transcriptional Repressor ZBTB4 Regulates EZH2 Through a MicroRNA-ZBTB4-Specificity Protein Signaling Axis. Neoplasia (New York, N.Y.), 16(12), 1059-1069.

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TaqMan SNP Genotyping Assay Protocol

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Three SNPs were analyzed using TaqMan^® Assays purchased from Thermo Fisher Scientific: rs6537958 (Assay ID: C_29220561_10), rs6537959 (Assay ID: C_29220562_20) and rs11103565 (Assay ID: C_31552275_10). For all three SNPs, the PCR mixture consisted of 10 µl of 2 × TaqMan™ Genotyping Master Mix (Thermo Fisher Scientific), 1 µl of appropriate 20X TaqMan^® Assay, and 10 ng of genomic DNA. The PCR mixture was filled up with a distilled, DNase and RNase free water (Gibco, Waltham, MA, USA) to the final volume of 20 µl. The real-time PCR was performed using the 7900HT Fast Real-Time PCR System (Applied Biosystems) with 96-Well Block Module using standard mode thermal cycling setting under the following conditions: an initial denaturation step at 95°C for 10 min, followed by 40 cycles of denaturation (95°C, 15 s), single annealing and extension step (60°C for 1 minute). Fluorescence signal detection for both dyes was performed after each cycle. For each plate, a no template control (NTC) was included. Allelic discrimination analysis was performed using a SDS 2.3 software (Applied Biosystems).

Świerzko A.S., Jarych D., Gajek G., Chojnacka K., Kobiela P., Kufelnicka-Babout M., Michalski M., Sobczuk K., Szala-Poździej A., Matsushita M., Mazela J., Domżalska-Popadiuk I., Kilpatrick D.C., Kalinka J., Sekine H, & Cedzyński M. (2021). Polymorphisms of the FCN2 Gene 3’UTR Region and Their Clinical Associations in Preterm Newborns. Frontiers in Immunology, 12, 741140.

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Quantitative Real-Time PCR Transcript Analysis

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Cell cultures (OD₇₅₀ = 0.40) corresponding to 2.2 × 10⁶ cells ml⁻¹ for IL, 5.5 × 10⁶ cells ml⁻¹ for A250R and 6.4 × 10⁶ cells ml⁻¹ for S264K, were harvested and total RNA extracted as previously described^{66 (link)}. Each RNA sample was subjected to DNase treatment using a Turbo DNA-free kit (Ambion). Total RNA (80 ng) was reverse transcribed and amplified using the SYBR Green PCR Master Mix and MuLV Reverse Transcriptase Reagents, according to one-step RT-PCR manufacturer’s protocol (Applied Biosystems, Foster city, USA). qRT-PCRs were performed in the Applied Biosystems 7900HT Real Time System with a standard 96-Well Block Module, using Frosted Subskirted optical tubes and Seal Film (Applied Biosystems, Foster city, USA). The reactions were subjected to a heat dissociation protocol using the 7900HT System software for melting curve analysis and detection of non-specific amplifications. A negative control without template was run with each assay to assess the overall specificity. The relative abundance of each gene was determined by the 2^−∆∆Ct method^{67 (link)}. RACK1 (receptor of activated protein kinase C1)^{68 (link)} was used as the endogenous control and for determining relative abundance of transcripts. Each assay included triplicate reactions. Primers were designed using Primer Quest (Integrated DNA Technologies, Coralville) (Table S5).

Antonacci A., Lambreva M.D., Margonelli A., Sobolev A.P., Pastorelli S., Bertalan I., Johanningmeier U., Sobolev V., Samish I., Edelman M., Havurinne V., Tyystjärvi E., Giardi M.T., Mattoo A.K, & Rea G. (2018). Photosystem-II D1 protein mutants of Chlamydomonas reinhardtii in relation to metabolic rewiring and remodelling of H-bond network at QB site. Scientific Reports, 8, 14745.

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