E coli dna polymerase 1

Manufactured by Thermo Fisher Scientific

Sourced in United States

E. coli DNA polymerase I is an enzyme that catalyzes the synthesis of DNA. It plays a crucial role in DNA replication, repair, and recombination processes in Escherichia coli.

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12 protocols using e coli dna polymerase 1

Transcriptome Analysis of PBMC Samples

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Total RNA was isolated from freshly purified PBMC stored in Trizol according to the manufacturer's instructions (Invitrogen) prior to reverse-transcription using a T7 oligo dT (24 (link)) primer and Superscript II (Invitrogen). Second-strand cDNA synthesis was obtained with RNase H, E. coli DNA polymerase I, and DNA ligase (Invitrogen). cDNA was blunt-ended with T4 DNA polymerase (Invitrogen) and purified using the QIAQuick PCR purification Kit™ (Qiagen, Germantown, MD). Labeling was performed using the ENZO BioArray™ HighYield™ RNA Transcript Kit (Affymetrix) according to the manufacturer's instructions. After purification, labeled cRNA was quantified by OD, and the quality was assessed on a Bioanalyzer (Agilent, Santa Clara, CA). Fragmented cRNA with hybridization controls and Oligo B2 from Affymetrix were hybridized on Affymetrix U133 Plus 2.0 Genechips for 18 hours followed by washing and staining on Affymetrix Fluidic 400. The arrays were scanned using the Affymetrix GeneChip Scanner 3000. Array images were analyzed using Affymetrix Gene Chip Operating System (GCOS) software using the MAS5 algorithm. The MAS5 algorithm (6 (link)) computes an adjusted signal level for each probe set to estimate expression of the target mRNA of that probe set. Values were normalized for each probe set across all arrays in the dataset.

Babu S.P., Chen Y.Y., Bonne-Annee S., Yang J., Maric I., Myers T.G., Nutman T.B, & Klion A.D. (2017). Dysregulation of interleukin 5 expression in familial eosinophilia. Allergy, 72(9), 1338-1345.

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Single-Cell RNA Amplification Protocol

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The product was subjected to linear T7-based antisense RNA (aRNA) amplification. Priming for second-strand cDNA synthesis was done by RNA nicking. Second-strand cDNA synthesis was carried out with E. coli DNA polymerase I (Invitrogen 18010025) followed by blunt-ending with T4 DNA polymerase to produce double-stranded cDNA that was purified with a MinElute PCR purification kit (Qiagen 28004). Linear T7-based in vitro aRNA synthesis from the sense strand was performed by T7 RNA polymerase using the Ambion MEGAscript T7 kit (Thermo Fisher AM1334). The RNA transcript reactions were cleaned using the Ambion Megaclear kit (Thermo Fisher AM1908) per the manufacturer’s instructions with some modifications, as described previously(Dueck et al., 2015 (link)). Synthesized aRNA was used in a second round of amplification for first-strand cDNA synthesis using random primers and second-strand cDNA synthesis with T7 promoter primer. This second round was repeated to achieve three rounds of amplification from each single cell. The RNA yield was quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop technologies).

Han L., Choudhury S., Mich-Basso J.D., Ammanamanchi N., Ganapathy B., Suresh S., Khaladkar M., Singh J., Maehr R., Zuppo D.A., Kim J., Eberwine J.H., Wyman S.K., Wu Y, & Kühn B. (2020). Lamin B2 levels regulate polyploidization of cardiomyocyte nuclei and myocardial regeneration. Developmental cell, 53(1), 42-59.e11.

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RNA-Seq Library Preparation Protocol

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Total RNA was extracted from each sample with an RNeasy Plant Mini kit (Qiagen), in which the first reagent of the kit (Buffer RLC) was added to the frozen tissue, and ground using a mortar and pestle. For each stage/tissue, 50 μg of total RNA were sent to the Joint Genome Institute (JGI). At JGI, mRNA was purified from total RNA using Absolutely mRNA™ purification kit (Stratagene) and chemically fragmented to 200-250bp (Ambion). mRNA was reverse transcribed with SuperScript II using random hexamers. Second strand cDNA was synthesized using dNTP/dUTP mix (Thermo Scientific), E. coli DNA Ligase, E. coli DNA polymerase I, and E coli RnaseH (Invitrogen). The fragments were treated with end-repair, A- tailing, and ligation of adaptors using the Illumina Truseq DNA Sample Prep Kit (Illumina). Second strand cDNA was removed by AmpErase UNG (Applied Biosystems) to generate strandedness similar to the method described by Parkhomchuk et al. [29 (link)] and enriched with 10 cycles of PCR to generate the final library. qPCR was used to determine the concentration of the libraries. Libraries were sequenced on the Illumina Hiseq, producing paired end reads R1 and R2 from each sample (fastq) with 100 bp in each read.

Muraguchi H., Umezawa K., Niikura M., Yoshida M., Kozaki T., Ishii K., Sakai K., Shimizu M., Nakahori K., Sakamoto Y., Choi C., Ngan C.Y., Lindquist E., Lipzen A., Tritt A., Haridas S., Barry K., Grigoriev I.V, & Pukkila P.J. (2015). Strand-Specific RNA-Seq Analyses of Fruiting Body Development in Coprinopsis cinerea. PLoS ONE, 10(10), e0141586.

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Second-strand synthesis and IVT protocol

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Next, 1,100 nl of second-strand synthesis mix (1.14× Second-Strand Buffer (Invitrogen), 0.23 mM (each) dNTP mix (Promega), 0.35 U of E. coli DNA Polymerase I (Invitrogen) and 20 mU of RNaseH (Invitrogen)) was added to each well. Plates were sealed and spun down at 2,000 r.c.f. for 2 minutes. Second-strand synthesis was carried out at 16 °C for 2 hours, followed by 85 °C for 20 minutes. Plates were snap-chilled, spun down at 2,000 r.c.f. for 1 minute and stored on ice before pooling. The protocol for pooling and in vitro transcription (IVT) was the same as SORT-seq^{27 (link)}.

Salmen F., De Jonghe J., Kaminski T.S., Alemany A., Parada G.E., Verity-Legg J., Yanagida A., Kohler T.N., Battich N., van den Brekel F., Ellermann A.L., Arias A.M., Nichols J., Hemberg M., Hollfelder F, & van Oudenaarden A. (2022). High-throughput total RNA sequencing in single cells using VASA-seq. Nature Biotechnology, 40(12), 1780-1793.

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Strand-Specific RNA-Seq Library Preparation

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RNA from human cell lines or mouse AML cells was isolated using the RNeasy Plus Mini Kit (Qiagen). mRNA was obtained using two rounds of poly(A) selection using the Dynabeads mRNA purification kit (Invitrogen) and subsequently fragmented by incubation at 94°C for 3 min (fragmentation buffer: 40 mM Tris base adjusted to pH 8.2 with glacial acetic acid, 100 mM potassium acetate, 30 mM magnesium acetate in DEPC-treated H₂O). Cell lines infected with shRNA expressing vectors were FAC-sorted for shRNA expression (GFP⁺) prior to RNA extraction. The fragmented mRNA was used as template for first-strand cDNA synthesis with random hexamers and a NEBNext DNA Library Prep Master Mix Set for Illumina Synthesis kit (Invitrogen). The second-strand cDNA was synthesized with 100 mM dATP, dCTP, dGTP and dUTP in the presence of RNase H, E. coli DNA polymerase I and DNA ligase (Invitrogen). The incorporation of dUTP allowed elimination of the second strand during library preparation (described below) and thus preservation of strand specificity.

Rathert P., Roth M., Neumann T., Muerdter F., Roe J.S., Muhar M., Deswal S., Cerny-Reiterer S., Peter B., Jude J., Hoffmann T., Boryń Ł.M., Axelsson E., Schweifer N., Tontsch-Grunt U., Dow L.E., Gianni D., Pearson M., Valent P., Stark A., Kraut N., Vakoc C.R, & Zuber J. (2015). Transcriptional plasticity promotes primary and acquired resistance to BET inhibition. Nature, 525(7570), 543-547.

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cDNA Library Preparation from Seed RNA

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Poly-A mRNA was purified from total RNA isolated from the June 5 and October 24 seeds using oligo (dT) magnetic beads and fragmented into 200–500 bp pieces using divalent cations. The mRNA fragments were reverse transcribed into first-strand cDNA using SuperScript II reverse transcriptase and random primers (Life Technologies). The second-strand cDNA was synthesized by E. coli DNA polymerase I (Invitrogen, USA). After double-stranded cDNA synthesis, fragments were end repaired and A-tailed. The final cDNA library was created by purifying and enriching the double-stranded cDNA with PCR.

Zeng Y., Tan X., Zhang L., Jiang N, & Cao H. (2014). Identification and Expression of Fructose-1,6-Bisphosphate Aldolase Genes and Their Relations to Oil Content in Developing Seeds of Tea Oil Tree (Camellia oleifera). PLoS ONE, 9(9), e107422.

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mRNA Fragmentation and cDNA Synthesis

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mRNA was fragmented and then reverse transcribed into cDNA. Zinc mediated fragmentation was performed by adding fragmentation reagent (Ambion, Applied Biosystems, Warrington, UK) to the mRNA and incubation at 70 °C for 5 min. First strand cDNA synthesis was performed using 3 μg of random hexamer primers and SuperScript II (Invitrogen). After the first strand was synthesized, second strand synthesis buffer, dNTPs, RNase H and E. coli DNA polymerase I (Invitrogen) were added and incubated for 2.5 h at 16 °C to translate the second-strand synthesis. DNA was then purified using a Qiaquick PCR spin column (Qiagen) and eluted in 30 μl EB buffer (Qiagen).

Matthews D., Diskin M.G., Kenny D.A., Creevey C.J., Keogh K, & Waters S.M. (2017). Effect of short term diet restriction on gene expression in the bovine hypothalamus using next generation RNA sequencing technology. BMC Genomics, 18, 857.

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Second Strand cDNA Synthesis Protocol

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In order to obtain the second strand of cDNA, the following were added to the mixture obtained after the first strand synthesis: 30 μL of 5x Second Strand Buffer, 91 μL of RNase-free water, 3 μL of 10 mM dNTPs, 4 μL (40 U) of E. coli DNA Polymerase I, 1 μL (10 U) of E. coli DNA Ligase, and 1 μL (2 U) of Rnase H (Invitrogen). The mixture was incubated for 2 hours at 16°C.
Next, 2.5 μL (10 U) of T4 DNA Polymerase I was added to the mixture and the mixture was incubated for another 5 minutes at 16°C. The composition of the reaction mixture used for the second strand cDNA synthesis is presented in Table 2. The reaction was stopped by adding 10 μL of 0.5 M EDTA, and the double-stranded cDNA was extracted using the phenol/chloroform/isoamyl alcohol mixture. The aqueous phase was divided by means of Phase Lock Gel Light 1.5 mL tubes (Eppendorf). 0.5 volume of 7.5 M ammonium acetate (Sigma) and 2.5 volume of 96% ethyl alcohol were added to the aqueous phase, and it was left for 12 h at −20°C.

Stojko R., Bojdys-Szyndlar M., Drosdzol-Cop A., Madej A, & Wilk K. (2015). Comparison of Signaling Pathways Gene Expression in CD34− Umbilical Cord Blood and Bone Marrow Stem Cells. Stem Cells International, 2016, 5395261.

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Immature B Cell RNA Sequencing

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RNA from ex vivo‐sorted immature B cells was isolated with the RNeasy Plus Mini Kit (Qiagen). mRNA was obtained by two rounds of poly(A) selection using the Dynabeads mRNA purification kit (Invitrogen) and fragmented by heating at 94°C for 3 min in fragmentation buffer. The fragmented mRNA was used as a template for first‐strand cDNA synthesis with random hexamers using the Superscript Vilo First‐Strand Synthesis System (Invitrogen). The second‐strand cDNA was synthesized with 100 mM dATP, dCTP, dGTP, and dUTP in the presence of RNase H, E. coli DNA polymerase I, and DNA ligase (Invitrogen).

Hill L., Jaritz M., Tagoh H., Schindler K., Kostanova‐Poliakova D., Sun Q., Schwickert T.A., Leeb M, & Busslinger M. (2023). Enhancers of the PAIR4 regulatory module promote distal VH gene recombination at the Igh locus. The EMBO Journal, 42(15), e112741.

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RNA-seq Library Preparation from MEF Cells

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One 15-cm tissue culture dish containing 1-2 x 10⁶ MEF cells was used for one RNA-seq experiment. The cells were processed for cDNA preparation as described26 (link). Cells were lysed using a QIAshredder column (Qiagen), and total RNA was extracted by the RNeasy Plus Mini kit (Invitrogen) using the gDNA Eliminator column. The purity of total RNA was analyzed with the Agilent RNA 6000 Nano kit (Agilent).
mRNA was purified in two rounds using the Dynabeads mRNA purification kit (Invitrogen), and the purified mRNA was fragmented for exactly 3 min at 94 °C in fragmentation buffer (8 mM Tris pH 8.2, 50 mM KAc and 4.5 mM MgAc) and thereafter purified over an RNeasy column. The quality of mRNA isolation and fragmentation was analyzed with the Agilent RNA 6000 Pico kit (Agilent). The fragmented mRNA was used as template for first-strand cDNA synthesis with random hexamers using the Superscript III First-Strand Synthesis kit (Invitrogen) followed by purification on a Mini Quick Spin column (Roche). The second-strand cDNA was synthesized by using random hexamers and 200 μM dATP, dCTP, dGTP and dUTP in the presence of RNase H, E. coli DNA polymerase I and DNA ligase (Invitrogen). The cDNA samples were purified with the MinElute Reaction Cleanup kit (Qiagen), and > 5 ng of cDNA were submitted for library preparation and Illumina deep sequencing to the Campus Science Support Facility.

Busslinger G.A., Stocsits R.R., van der Lelij P., Axelsson E., Tedeschi A., Galjart N, & Peters J.M. (2017). Cohesin is positioned in mammalian genomes by transcription, CTCF and Wapl. Nature, 544(7651), 503-507.

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