![]() Serebrov V, Vassilenko K, Kholod N, Gross HJ, Kisselev L (1998) Mg2+ binding and structural stability of mature and in vitro synthesized unmodified Escherichia coli tRNAPhe. DNA Pol I, Large (Klenow) fragment was originally derived as a proteolytic. Klein DJ, Moore PB, Steitz TA (2004) The contribution of metal ions to the structural stability of the large ribosomal subunit. NEB2 cloned at NEB recombinant 37 75 Heat. Protocol for blunting ends by 3' overhang removal and fill-in of 3' recessed (5' overhang) ends using DNA Polymerase I, Large (Klenow) Fragment (M0210) Protocols.io also provides an interactive version of this protocol where you can discover and share optimizations with the research community. ![]() Hall KB, Sampson JR, Uhlenbeck OC, Redfield AG (1989) Structure of an unmodified tRNA molecule. Gholamalipour Y, Karunanayake Mudiyanselage A, Martin CT (2018) 3′ end additions by T7 RNA polymerase are RNA self-templated, distributive and diverse in character-RNA-Seq analyses. Wurtmann EJ, Wolin SL (2009) RNA under attack: cellular handling of RNA damage. Milligan JF, Groebe DR, Witherell GW, Uhlenbeck OC (1987) Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. It retains polymerase activity, but lacks both 5’ > 3’ and 3' > 5’ exonuclease activity. Īrnaud-Barbe N, Cheynet-Sauvion V, Oriol G, Mandrand B, Mallet F (1998) Transcription of RNA templates by T7 RNA polymerase. DNA Pol I, Large (Klenow) fragment was originally derived as a proteolytic product of E. All fragments have a 4-base, 5´ overhangs that can be end labeled using T4 Polynucleotide Kinase (M0201) or filled-in using DNA Polymerase I, Klenow Fragment (M0210) (1). Imburgio D, Rong M, Ma K, McAllister WT (2000) Studies of promoter recognition and start site selection by T7 RNA polymerase using a comprehensive collection of promoter variants. Ĭonrad T, Plumbom I, Alcobendas M, Vidal R, Sauer S (2020) Maximizing transcription of nucleic acids with efficient T7 promoters. Libraries were quantified with the Bioanalyzer 2100 (Agilent) and sequenced on the Illumina HiSeq 2000 Sequencer (100 nucleotide read length).Chan PP, Lowe TM (2016) GtRNAdb 2.0: an expanded database of transfer RNA genes identified in complete and draft genomes. When using Mung Bean Nuclease (NEB M0250), incubate the reaction at room temperature. PCR products were size selected using 3% NuSieve agarose gels (Lonza) followed by gel extraction using QIAEX II reagents (QIAGEN). Make sure to add a sufficient amount of dNTPs to the reaction (33 M each dNTP for DNA Polymerase I, Large (Klenow) Fragment, NEB M0210 and 100 M each dNTP for T4 DNA Polymerase, NEB M0203). The adaptor-modified DNA fragments were enriched by PCR using the Illumina Barcode primers and Phusion DNA polymerase (NEB). A single A-base was added to fragment ends by Klenow fragment (3' to 5' exo minus NEB) followed by ligation of Illumina adaptors (Quick ligase, NEB). ChIP-enriched DNA samples (1-10 ng) were converted to blunt-ended fragments using T4 DNA polymerase, E.coli DNA polymerase I large fragment (Klenow polymerase) and T4 polynuleotide kinase (New England BioLabs, NEB). The ChIP-seq sample preparation for sequencing was performed according to the manufacturer's instructions (Illumina). These samples were then immunoblotted as described above with the exception of using protein A-HRP secondary (GE HealthCare, Cat#: NA9120-1ML) antibody for detection. Beads were washed 3 times with IP buffer (150nM NaCl Thermo Fisher Scientific) and directly boiled in 1X NuPage LDS/reducing agent buffer (ThermoFisher Scientific Cat#: NP0007 and NP0009) to elute and denature the precipitated proteins. Note: Klenow Fragment, Exonuclease Minus, will leave a single-base 3´-overhang for a significant proportion of the DNA fragments during the fill-in reaction (8). Stop the reaction by heating the mixture for 10 minutes at 75☌. Incubate the reaction at room temperature for 10 minutes. A temperature gradient can also be used to optimize the annealing temperature for each primer pair. Typically, use a 1030 second annealing step at 3☌ above the Tm of the lower Tm primer. Next day, 50ul of Dynabeads Protein-G beads were added to the lysate-antibody mixture and incubated for 2h at 4C. unit of Klenow Fragment per microgram of DNA. The NEB Tm Calculator should be used to determine the annealing temperature when this enzyme. A fraction of the cleared lysate was saved as input and the remainder was incubated overnight (12-16 hours) with 10ug of target protein antibody at 4C with gentle mixing. Nuclear lysates were incubated for 2 hours at 4C with 30ul of magnetic Protein-G Dynabeads (Thermo Fisher Scientific Cat#: 10004D) for pre-clearing. Library_strategy ChIP-Seq library_source GENOMIC library_selection ChIP library_construction_protocol 8-10 million cells ectopically overexpressing different V5-tagged FOXA1 variants and WT AR (or TLE3) were fractionated to isolated intact nuclei using the NE-PER kit reagents (Thermo Fisher Scientific Cat#: 78835) and lysed in the complete IP lysis buffer (Thermo Fisher Scientific Cat#: 87788).
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