NEBuffers r1.1 and 1 are not recommended when overhang removal is required. Heat the tubes on a Thermomixer at 55☌ for 2 min with mixing. 51) Wash the beads by adding 600l of 1X TWB and transferring the mixture to a new tube. Separate on a magnet and discard the solution. 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. For blunting reactions requiring removal of overhangs: DNA Polymerase I, Large (Klenow) Fragment can be used in NEBuffers r2.1, r3.1, and rCutSmart Buffer as well as NEBuffers 2, 3, and 4 and T4 DNA Ligase Buffer. 5l of 5U/l NEB Klenow exo minus (NEB, M0212) 50) Incubate at 37☌ for 30 minutes. Klein DJ, Moore PB, Steitz TA (2004) The contribution of metal ions to the structural stability of the large ribosomal subunit.
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. Īrnaud-Barbe N, Cheynet-Sauvion V, Oriol G, Mandrand B, Mallet F (1998) Transcription of RNA templates by T7 RNA polymerase. 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. Removal of Single-Stranded Extension Protocol using Mung Bean Nuclease (M0250) Optimizing Restriction Endonuclease Reactions. Ĭonrad T, Plumbom I, Alcobendas M, Vidal R, Sauer S (2020) Maximizing transcription of nucleic acids with efficient T7 promoters. The cloned gene for Pol I has also been modified to overproduce the Klenow fragment. Some commercial Pol Ik are produced by the proteolytic digestion of the purified, cloned Pol I. 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. The Klenow fragment was originally produced by limited proteolysis of Pol I using a bacterial protease, subtilisin, at pH 6.5 in K-P buffer (102 ). PCR products were size selected using 3% NuSieve agarose gels (Lonza) followed by gel extraction using QIAEX II reagents (QIAGEN). 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). DNA Polymerase I, Large (Klenow) Fragment ( NEB M0210 ), T4 DNA Polymerase ( NEB M0203 ) or Mung Bean Nuclease ( NEB M0250) are the best choices to remove 3' overhangs. To create blunt ends 3' overhangs must be removed. DNA Polymerase I, Large (Klenow) Fragment retains polymerase and 3' to 5' exonuclease activity, but lacks 5' to 3' exonuclease activity. 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. No, DNA polymerases cannot fill in 3' overhangs.
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. Next day, 50ul of Dynabeads Protein-G beads were added to the lysate-antibody mixture and incubated for 2h at 4C. 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).
0 kommentar(er)
