Dna polymerase 1

Manufactured by Qiagen

DNA polymerase I is an enzyme responsible for the replication and repair of DNA. It catalyzes the synthesis of new DNA strands complementary to a template DNA strand.

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Lab products found in correlation

Superscript 3, by Thermo Fisher Scientific (2 mentions) Ribominus, by Thermo Fisher Scientific (1 mentions) Rna fragmentation reagent, by Thermo Fisher Scientific (1 mentions) Superscript 2, by Thermo Fisher Scientific (1 mentions) Pcr purification kit, by Qiagen (1 mentions) Hiseq 2000, by Illumina (1 mentions) Bioanalyzer, by Agilent Technologies (1 mentions) Mirneasy, by Qiagen (1 mentions) Dynabeads oligo dt 25, by Thermo Fisher Scientific (1 mentions) Quick t4 dna ligase, by New England Biolabs (1 mentions) Uracil dna glycosylase, by Qiagen (1 mentions) Nebnext high fidelity pcr master mix, by New England Biolabs (1 mentions)

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DNA polymerase (4 citations)

3 protocols using dna polymerase 1

Transcriptome Analysis of Tumor Samples

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Total RNA was isolated from tumors (miRNeasy, Qiagen) and validated to have a median RNA Integrity Numbers (RIN) of 8.6 (minimum 6.8) using a Bioanalyzer (Agilent). Ribosomal RNA was depleted (RiboMinus, Invitrogen) and RNA was fragmented (RNA Fragmentation Reagents, Ambion). cDNA was generated (SuperScript II, Invitrogen) by random priming followed by second strand synthesis (DNA polymerase I, Enzymatics) and purified (PCR purification kit, Qiagen). Libraries were prepared according to the manufacturer's specifications (Illumina). Sequencing was performed using 50-bp single-end reads (Illumina HiSeq 2000). Reads were aligned to the reference human genome (hg19) using TopHat (Trapnell et al. 2009 (link)), and gene expression was estimated by calculating RPKM, analyzing only exonic reads. Intron retention scores were calculated for each gene as follows:

Simon J.M., Hacker K.E., Singh D., Brannon A.R., Parker J.S., Weiser M., Ho T.H., Kuan P.F., Jonasch E., Furey T.S., Prins J.F., Lieb J.D., Rathmell W.K, & Davis I.J. (2014). Variation in chromatin accessibility in human kidney cancer links H3K36 methyltransferase loss with widespread RNA processing defects. Genome Research, 24(2), 241-250.

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RNA-Seq Library Preparation Protocol

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RNA-Seq libraries were prepared as described by Zhong et al.57 (link) with minor modifications. Briefly, 5 μg of total RNA was used for poly(A) RNA capture using Dynabeads Oligo (dT)₂₅ (Invitrogen), fragmented at 94 °C for 5 minutes and eluted. The first-strand cDNA was synthesized using reverse transcriptase SuperScript III (Invitrogen) with random primers and dNTP, whereas the second-strand cDNA was generated using DNA polymerase I (Enzymatics) using dUTP. After end-repair (Enzymatics), dA-tailing with Klenow 3′-5′ (Enzymatics) and adapter ligation (Quick T4 DNA Ligase, NEB), the dUTP-containing second-strand was digested by uracil DNA glycosylase (Enzymatics). The resulting first-strand adaptor-ligated cDNA was used for PCR enrichment (NEBNext High-Fidelity PCR Master Mix, NEB) for 14 cycles. Indexed libraries were pooled and sequenced.

Cárdenas P.D., Sonawane P.D., Pollier J., Vanden Bossche R., Dewangan V., Weithorn E., Tal L., Meir S., Rogachev I., Malitsky S., Giri A.P., Goossens A., Burdman S, & Aharoni A. (2016). GAME9 regulates the biosynthesis of steroidal alkaloids and upstream isoprenoids in the plant mevalonate pathway. Nature Communications, 7, 10654.

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Microglia RNA Extraction and Sequencing

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Isolated microglia were pelleted and put into 150 µl lysis/Oligo d(T) Magnetic Beads binding buffer and stored at −80°C until processing. For human brain cortical RNA, 500 ng of RNA was diluted with proper volume of 2× lysis/ Oligo d(T) Magnetic Beads binding buffer to a final concentration of 1× lysis/Oligo d(T) Magnetic Beads binding buffer. mRNAs were enriched by incubation with Oligo d(T) Magnetic Beads (NEB, S1419S) and then fragmented/eluted by incubation at 94 °C for 9 min. cDNA was then synthesized with Superscript III (first-strand synthesis; Life Technologies kit 18080-044) and then DNA Polymerase I (second-strand synthesis; Enzymatics P7050L)

Gosselin D., Skola D., Coufal N.G., Holtman I.R., Schlachetzki J.C., Sajti E., Jaeger B.N., O’Connor C., Fitzpatrick C., Pasillas M.P., Pena M., Adair A., Gonda D.G., Levy M.L., Ransohoff R.M., Gage F.H, & Glass C.K. (2017). An environment-dependent transcriptional network specifies human microglia identity. Science (New York, N.Y.), 356(6344), eaal3222.

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