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C1181

Manufactured by Promega
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The C1181 is a centrifuge from Promega designed for general laboratory use. It is a versatile and reliable equipment that can be used for a variety of common laboratory procedures that require centrifugation.

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Lab products found in correlation

Steponeplus real time pcr machine, by Thermo Fisher Scientific (5 mentions) Trizol reagent, by Thermo Fisher Scientific (5 mentions) Power sybr green pcr master mix, by Thermo Fisher Scientific (5 mentions) M mulv reverse transcriptase, by New England Biolabs (3 mentions) Cell recovery solution, by Corning (2 mentions) Rnase inhibitor m0314l, by New England Biolabs (2 mentions) Dntps n0447s, by New England Biolabs (2 mentions) Moloney murine leukemia virus reverse transcriptase, by Promega (1 mentions) Rnasin, by Promega (1 mentions) Rneasy kit, by Qiagen (1 mentions) Quick rna extraction kit, by Zymo Research (1 mentions) Corbett rotorgene 600 instrument, by Qiagen (1 mentions) Total rna mini kit, by Geneaid (1 mentions) M1701, by Promega (1 mentions) Steponeplus machine, by Thermo Fisher Scientific (1 mentions) Perfecta sybr green supermix, by Thermo Fisher Scientific (1 mentions) Rneasy mini kit, by Qiagen (1 mentions) Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (1 mentions) Qubit rna broad range assay, by Thermo Fisher Scientific (1 mentions) Sybr green pcr master mix, by Promega (1 mentions)

12 protocols using c1181

1

RNA Extraction from 3D Matrigel-Cultured mES Cells

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For RNA extraction of mES cells cultured in Matrigel (3D on top
protocol), the Matrigel was first removed by treatment with Cell Recovery
Solution (354253, Corning) for 15 min at 4 °C. To facilitate the
depolimerization, the Matrigel was manually broken into small pieces. Cells were
subsequently centrifuged and pellets were washed once with PBS.
RNA was extracted using TRIzol reagent (15596010, Thermo Fisher
Scientific) following the manufacturer’s instructions. Subsequently, 1
μg of RNA was used to perform a reverse transcriptase reaction in the
presence of random primers (C1181, Promega), dNTPs (N0447S, New England
BioLabs), RNase inhibitor (M0314L, New England BioLabs) and M-MuLV reverse
transcriptase (M0253L, New England BioLabs). RT–PCR reactions were
carried out using Power SYBR Green PCR Master Mix (4368708, ThermoFisher
Scientific) on a Step One Plus Real-Time PCR machine (Applied Biosystems). The
following program was used: 10 min 95 °C followed by 40 cycles of 15 s 95
°C (denaturing) and 1 min 60 °C (annealing and extension). The
primers used are listed in Supplementary Table 3.
Mouse gene expression data were normalized to Gapdh;
human gene expression data were normalized to HPRT1. Error bars
represent variability (s.e.m.) across multiple independent experiments.
Shahbazi M.N., Scialdone A., Skorupska N., Weberling A., Recher G., Zhu M., Jedrusik A., Devito L.G., Noli L., Macaulay I.C., Buecker C., Khalaf Y., Ilic D., Voet T., Marioni J.C, & Zernicka-Goetz M. (2017). Pluripotent state transitions coordinate morphogenesis in mouse and human embryos. Nature, 552(7684), 239-243.
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2

Quantification of mRNA Levels by qPCR

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Total cellular RNA was extracted using TRIzol reagent (Life Technologies) and cDNA was synthesized with 200 U of Moloney murine leukemia virus-reverse transcriptase (Promega), 5 pmol of random primers (C1181, Promega), 40 U RNAsin (Promega), and 2.5 mM deoxynucleotide triphosphate. mRNA levels were quantitated on a Mastercycler ep Realplex (MasterCycler2, Eppendorf, Le Pecq, France) with Mesagreen QPCR Mastermix Plus for SYBR (Promega). Sequences of the primers are the following: NPM1, 5′-ATGGAAGATTCGATGGACATGG-3′ (Forward) and 5′-CGAGAAGAGACTTCCTCCACTGC -3′ (Reverse); PCNA, 5′-TGCCTTCTGGTGAATTTGCACGT-3′ (Forward) and 5′- ACCGTTGAAGAGAGTGGAGTGGC-3′ (Reverse), Actin, 5′-CGCGAGAAGATGACCCAGATC-3′ (Forward) and 5′TCACCGGAGTCCATCACGA-3′ (Reverse) (Eurogentec, Angers, France). For human EGF, primers were purchased from Qiagen (PPH00137B-200, Qiagen, Courtaboeuf, France).
Loubeau G., Boudra R., Maquaire S., Lours-Calet C., Beaudoin C., Verrelle P, & Morel L. (2014). NPM1 Silencing Reduces Tumour Growth and MAPK Signalling in Prostate Cancer Cells. PLoS ONE, 9(5), e96293.
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3

Quantitative RT-PCR Gene Expression Analysis

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Cell pellets were collected and RNA was extracted using the Qiagen RNeasy kit following the manufacturer’s instructions. The reverse transcriptase reaction was performed with 1 µg RNA with random primers (C1181, Promega), dNTPs (N0447S, New England BioLabs), RNAse inhibitor (M0314L, New England Biolabs, and M-MuLV reverse transcriptase (M0253L, New England Biolabs). Quantitative PCR with reverse transcription was performed using Power SYBR Green PCR Master Mix (4368708, Thermo Fisher Scientific) on a Step One Plus Real-Time PCR machine (Applied Biosystems). The following program was used: 10 min at 95 °C followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C. Single melt curves were observed for all primers used in this study. Oligonucleotides used in this study are provided in Supplementary Table 4.
Weatherbee B.A., Gantner C.W., Iwamoto-Stohl L.K., Daza R.M., Hamazaki N., Shendure J, & Zernicka-Goetz M. (2023). Pluripotent stem cell-derived model of the post-implantation human embryo. Nature, 622(7983), 584-593.
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4

Molecular Profiling of Engineered Tissues

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Total RNA was extracted from engineered tissues resulting from the 5 weeks in vitro culture using the Quick-RNA™ extraction kit (Zymo Research, Irvine, CA, USA, R1055), according to manufacturer’s instructions. Following cDNA synthesis (Invitrogen 18080044 & Promega, C1181), qRT-PCR was performed using assay on demand (Applied Biosystems, Foster City, CA, USA) on the following genes: Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Hs02758991_g1), Indian Hedge Hog (IHH, Hs01081800_m1), SRY-box 9 (SOX9, Hs00165814_m1), Matrix metalloproteinase 13 (MMP13, Hs00233992_m1), Collagen type II (ColII, Hs00264051_m1), Collagen type X (ColX, Hs00166657_m1), Vascular endothelial growth factor (VEGF, Hs00900055_m1), Bone morphogenetic protein 2 (BMP2, Hs00154192), Osteocalcin (OCN, Hs01587814_g1), and Bone sialoprotein (BSP, Hs00959010_m1).
Pigeot S., Bourgine P.E., Claude J., Scotti C., Papadimitropoulos A., Todorov A., Epple C., Peretti G.M, & Martin I. (2020). Orthotopic Bone Formation by Streamlined Engineering and Devitalization of Human Hypertrophic Cartilage. International Journal of Molecular Sciences, 21(19), 7233.
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5

RNA Extraction and RT-PCR Analysis

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RNA was extracted using TRIzol reagent (15596010, Thermo Fisher Scientific) following the manufacturer’s instructions. The reverse transcriptase reaction was performed with 1 μg of RNA in the presence of random primers (C1181, Promega), dNTPs (N0447S, New England BioLabs), RNAse inhibitor (M0314L, New England Biolabs) and M-MuLV reverse transcriptase (M0253L, New England BioLabs). RT-PCR reactions were carried out on a Step One Plus Real-Time PCR machine (Applied Biosystems) using Power SYBR Green PCR Master Mix (4368708, Thermo Fisher Scientific). A list of all the primers used is provided in Supplementary Table 3. The following program was used: 10 min 95 °C denaturation and 40 cycles of 15 s 95 °C and 1 min 60 °C.
Shahbazi M.N., Wang T., Tao X., Weatherbee B.A., Sun L., Zhan Y., Keller L., Smith G.D., Pellicer A., Scott RT J.r., Seli E, & Zernicka-Goetz M. (2020). Developmental potential of aneuploid human embryos cultured beyond implantation. Nature Communications, 11, 3987.
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6

RNA Extraction from 3D Matrigel-Cultured mES Cells

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For RNA extraction of mES cells cultured in Matrigel (3D on top
protocol), the Matrigel was first removed by treatment with Cell Recovery
Solution (354253, Corning) for 15 min at 4 °C. To facilitate the
depolimerization, the Matrigel was manually broken into small pieces. Cells were
subsequently centrifuged and pellets were washed once with PBS.
RNA was extracted using TRIzol reagent (15596010, Thermo Fisher
Scientific) following the manufacturer’s instructions. Subsequently, 1
μg of RNA was used to perform a reverse transcriptase reaction in the
presence of random primers (C1181, Promega), dNTPs (N0447S, New England
BioLabs), RNase inhibitor (M0314L, New England BioLabs) and M-MuLV reverse
transcriptase (M0253L, New England BioLabs). RT–PCR reactions were
carried out using Power SYBR Green PCR Master Mix (4368708, ThermoFisher
Scientific) on a Step One Plus Real-Time PCR machine (Applied Biosystems). The
following program was used: 10 min 95 °C followed by 40 cycles of 15 s 95
°C (denaturing) and 1 min 60 °C (annealing and extension). The
primers used are listed in Supplementary Table 3.
Mouse gene expression data were normalized to Gapdh;
human gene expression data were normalized to HPRT1. Error bars
represent variability (s.e.m.) across multiple independent experiments.
Shahbazi M.N., Scialdone A., Skorupska N., Weberling A., Recher G., Zhu M., Jedrusik A., Devito L.G., Noli L., Macaulay I.C., Buecker C., Khalaf Y., Ilic D., Voet T., Marioni J.C, & Zernicka-Goetz M. (2017). Pluripotent state transitions coordinate morphogenesis in mouse and human embryos. Nature, 552(7684), 239-243.
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7

RNA Extraction and Gene Expression Analysis

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RNA was extracted from tissues and LβT2 cells using TRIzol reagent (15596018, Invitrogen, Waltham, MA, USA) following the manufacturer’s protocol. For primary pituitary cultures, RNA was extracted using the Total RNA Mini Kit (FA32808-PS, Geneaid) following the manufacturer’s protocol. For the assessment of Tgfbr3l expression in different tissues and in LβT2 cells, 1 μg of total RNA was reverse-transcribed. For all other experiments, 200 ng (tissue) or 100 ng (primary culture) of total RNA was reverse-transcribed.
RNA was reverse-transcribed using random hexamers (C1181, Promega) and Moloney murine leukemia virus reverse transcriptase (M1701, Promega). The resulting cDNA was used for qPCR analysis using EvaGreen (ABMMmix, Diamed, Missisauga, ON, Canada) and primers listed in table S2 on a Corbett Rotorgene 600 instrument (Corbett Life Science, Sydney, NSW, Australia). mRNA levels were determined using the 2−ΔΔCT method. Gene expression was normalized to ribosomal protein L19 (Rpl19). All primers were validated for efficiency and specificity.
Brûlé E., Wang Y., Li Y., Lin Y.F., Zhou X., Ongaro L., Alonso C.A., Buddle E.R., Schneyer A.L., Byeon C.H., Hinck C.S., Mendelev N., Russell J.P., Cowan M., Boehm U., Ruf-Zamojski F., Zamojski M., Andoniadou C.L., Sealfon S.C., Harrison C.A., Walton K.L., Hinck A.P, & Bernard D.J. (2021). TGFBR3L is an inhibin B co-receptor that regulates female fertility. Science Advances, 7(51), eabl4391.
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8

Modified DNA Extraction from Mars Analog

Cited 1 time
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To parallel the unmodified extractions, an estimated 1.6 × 104 spores of B. subtilis and vegetative cells of E. coli were deposited on 50 mg of Mars analog soil, respectively. All samples were processed by using the “near-universal” modified extraction protocol: (1) Suspend sample in 800 μL of 8 × binding buffer and vortex gently for 30 s, (2) desalt in a single 100K Amincon Ultra column (Z740183), (3) resuspend the sample in 400 μL of molecular-grade water and 400 μL of 8 × binding buffer, (4) add 4–6 μg of random hexamer primers (Promega, C1181) and vortex gently for 30 s, (5) lyse cells and bind DNA with Purelyse at 6.5 V for 2 min, (6) wash with 2.5 mL of 1 × binding buffer at 1.5 V for 1 min, (7) elute DNA with 200 μL of heated elution buffer to 70°C for 1 min, (8) repeat step 7 for second elution. Basalt and alkaline samples contained 4 μg of random hexamer primers as we observed a decrease in DNA yield with increasing amounts. We believe this is most likely due to hexamer-induced competitive binding since basalt and alkaline samples have a lower DNA binding affinity (Mojarro et al.,2017b (link)). Meanwhile JSC, acid, salt, aeolian, and perchlorate samples required 6 μg.
Mojarro A., Hachey J., Bailey R., Brown M., Doebler R., Ruvkun G., Zuber M.T, & Carr C.E. (2019). Nucleic Acid Extraction and Sequencing from Low-Biomass Synthetic Mars Analog Soils for In Situ Life Detection. Astrobiology, 19(9), 1139-1152.
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9

Reverse Transcription and qPCR Analysis

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To make sample cDNA, 1 μg of RNA was used to perform a reverse transcriptase reaction in the presence of random primers (C1181, Promega), dNTPs (N0447S, New England BioLabs), RNase inhibitor (M0314L, New England BioLabs), and M-MuLV reverse transcriptase (M0253L, New England BioLabs).
RT-PCR reactions were carried out using Power SYBR Green PCR Master Mix (4368708, Thermo Fisher Scientific) on a Step One Plus Real-Time PCR machine (Applied Biosystems). The following programme was used: 10 min at 95°C and 40 cycles of 15 s at 95°C and 1 min at 60°C. qPCR data was normalised against expression levels of hypoxanthine guanine phosphoribosyltransferase (HPRT), which should be consistent across samples. Error bars represent variability (SEM) across multiple independent experiments. A complete list of primers used is shown in Table S4.
Mackinlay K.M., Weatherbee B.A., Souza Rosa V., Handford C.E., Hudson G., Coorens T., Pereira L.V., Behjati S., Vallier L., Shahbazi M.N, & Zernicka-Goetz M. (2021). An in vitro stem cell model of human epiblast and yolk sac interaction. eLife, 10, e63930.
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10

Quantification of RNA Binding Proteins

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RNA was isolated from biological triplicate (cells were grown overnight in separate flasks before harvesting) samples. Equivalent amounts of RNA from input and IP samples (∼350 ng) were used to produce cDNA using random hexadeoxynucleotide primers ([final] = 0.01 μg/μl, Promega #C1181) and AMV reverse transcriptase (0.4 unit/µl) for 1 hr at 42° in a 15 μl volume. Real-time PCR was performed using Quanta PerfeCTa SYBR Green SuperMix in a 96-well plate (in technical triplicate) in an Applied Biosystems StepOnePlus machine. For each input and IP, the average Ct value was calculated from the technical triplicates. Then the %IP was calculated using the equation 2^(CtInput(avg)−CtIP(avg)). The average and SD of three biological replicates was reported using the AVERAGE and STDEV functions in Microsoft Excel. See File S5 for a list of primers.
Miller J.E., Zhang L., Jiang H., Li Y., Pugh B.F, & Reese J.C. (2017). Genome-Wide Mapping of Decay Factor–mRNA Interactions in Yeast Identifies Nutrient-Responsive Transcripts as Targets of the Deadenylase Ccr4. G3: Genes|Genomes|Genetics, 8(1), 315-330.
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