Fosmid probes WIBR1-0935O10 (Nkx2.3 mm9; chr19; 43,659,682-43,698,592), WIBR1-1122P14 (Pax2 mm9; chr19; 44,809,035-44,851,675), WIBR1-1125H10 (Dlx2 mm9; chr2: 71374041-71411685), WIBR1-2777G14 (HoxD10 mm9; chr2: 74511607-74550498) were obtained from BACPAC Resources Center (Children’s Hospital Oakland Research Institute; [https://bacpacresources.org/ ]). Probes were labeled for use in FISH by nick translation as follows: prior to nick translation, 1 μg DNA was treated with RNase (0.02 U) (Sigma), for 30 min at 37°C, nick translation was carried out at 16°C for 1 h in the following reaction mixture; 50 mM Tris-HCl, 5 mM MgCl2, 2.5 μg BSA, 10 mM β-mercaptoethanol, 50 mM dAGC, 20 μM hapten/fluor [digoxigenin-11-dUTP (Sigma); Cy3 dUTP (GE Healthcare)], 15 U recombinant DNase1 (Sigma) and 10 U DNA polymerase I (NEB), made up to a final volume of 50 μL with H20.
Dna polymerase 1
Manufactured by Merck Group
DNA polymerase I is an enzyme that catalyzes the synthesis of DNA from deoxyribonucleotide precursors. It is responsible for the replication and repair of DNA in many organisms.
Automatically generated - may contain errors
Lab products found in correlation
Dntps, by Merck Group (2 mentions)
Rnase h, by Merck Group (2 mentions)
Digoxigenin 11 dutp, by Merck Group (1 mentions)
Cy3 dutp, by GE Healthcare (1 mentions)
Qubit 2.0 fluorometer, by Illumina (1 mentions)
Superscript 3, by Thermo Fisher Scientific (1 mentions)
Hiseq 4000 platform, by Illumina (1 mentions)
Bglii, by New England Biolabs (1 mentions)
Genelute gel extraction kit, by Merck Group (1 mentions)
Quick ligation kit, by New England Biolabs (1 mentions)
Dreamtaq green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Eurogold plasmid miniprep kit, by Euroclone (1 mentions)
Ecori hf, by New England Biolabs (1 mentions)
Genelute bacterial genomic dna kit, by Merck Group (1 mentions)
Bamhi hf, by New England Biolabs (1 mentions)
Xbai, by New England Biolabs (1 mentions)
Pcrbio hifi polymerase, by PCR Biosystems (1 mentions)
Steponeplus real time pcr system, by Illumina (1 mentions)
Solid whole transcriptome analysis kit, by Thermo Fisher Scientific (1 mentions)
Hiseq 2000 sequencer, by Illumina (1 mentions)
4 protocols using dna polymerase 1
1
Fosmid Probe Labeling for FISH
2
Illumina RNA-Seq Library Preparation
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Before Illumina sequencing, all six RNA samples that possessed poly(A) were enriched with oligo (dT) magnetic beads. The enriched RNA was randomly reduced to small pieces with a fragmentation buffer. First strand cDNA was generated using hexamers and reverse transcriptase (Superscript III, Invitrogen). After purification with AMPure XP beads, second strand cDNA was synthesized using DNA polymerase I, RNase H and dNTPs (Sigma-Aldrich). The double-stranded cDNA was subjected to terminal repair and poly(A) tailing, followed by Illumina adaptor ligation. The final cDNA library was completed after a second round of purification and PCR amplification. The quality of the six cDNA libraries was assessed using a Qubit 2.0 fluorometer prior to sequencing on the Illumina HiSeq 4000 platform.
3
Genetic Engineering Protocols for DNA Manipulation
4
RNA-Seq Library Preparation and Sequencing
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The following analysis was executed by KangChen Biotech. In accordance with their experimental procedures, a random fragment sequencing library was constructed using a SOLiD Whole Transcriptome Analysis Kit (Life Technologies). Nucleic acid cleaving reagents were added, and the mRNA was randomly disrupted into short segments in a shaking incubator. First‐strand cDNA was reverse transcribed using the fragmented mRNA as the template; and second‐strand cDNA was synthesized using a second‐strand DNA‐synthesis reaction system consisting of DNA polymerase I, dNTPs, and RNase H (Sigma). The synthesized DNA was purified using a DNA purification kit and recovered. The base ‘A’ was added to the 3’end of the cDNA, followed by ligation to the adapter in order to complete the blunt‐end repair reaction. Subsequently, we performed DNA fragment‐size selection. Finally, the cDNA was used for PCR amplification to obtain a sequencing library. The constructed library was qualified using an Agilent 2100 Bioanalyzer and the ABI StepOnePlus Real‐Time PCR System, and it was subjected to high‐throughput sequencing using an Illumina HiSeq™ 2000 Sequencer after passing quality controls.
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