binding buffer  (New England Biolabs)

Journal: Nature

Article Title: Poly(A)-tail profiling reveals an embryonic switch in translational control

doi: 10.1038/nature13007

Figure Lengend Snippet: Global measurement of poly(A)-tail lengths a , Outline of PAL-seq. For each cluster, the fluorescence intensity reflects the tail length of the cDNA that seeded the cluster. Although the probability of incorporating a biotin-conjugated dU opposite each tail nucleotide is uniform, stochastic incorporation results in a variable number of biotins for each molecule within a cluster. b , Median streptavidin fluorescence intensities for two sets of mRNA-like molecules with indicated poly(A)-tail lengths, which were added to 3T3 (circle), HEK293T (triangle), and HeLa (square) samples for tail-length calibration.

Article Snippet: After three additional cycles of cleavage to remove any residual sequencing fluorophores, the flow cell was washed with buffer [40.25 mM phosphate buffered saline (PBS), pH 7.4, 0.1% Tween], blocked with streptavidin-binding buffer [300 µg/ml bovine serum albumin (NEB), 40.25 mM PBS, pH 7.4, 0.1% Tween], washed with buffer again, and then imaged, as done previously .

Techniques: Fluorescence

Journal: Nucleic Acids Research

Article Title: Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis

doi: 10.1093/nar/gky067

Figure Lengend Snippet: Darwin Assembly using a θ oligonucleotide. Here, a single θ oligonucleotide is used in place of the two boundary oligonucleotides allowing enzymatic cleanup after the assembly reaction. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the nicked strand degraded by exonuclease III (1). Inner oligonucleotides and a single θ oligonucleotide are annealed to the ssDNA plasmid (2). The θ oligonucleotide encodes both assembly priming and termination sequences linked by a flexible linker such that successful assembly of the mutated strand results in a closed circle (3). The template plasmid can now be linearized (e.g. at the yellow dot, by adding a targeting oligonucleotide and appropriate restriction endonuclease) and both exonuclease I and exonuclease III added to degrade any non-circular DNA (4). The mutated gene can now be amplified from the closed circle by PCR (5) and cloned into a fresh vector (6) using the type IIS restriction sites (white dots).

Article Snippet: PCR products were purified using GeneJET PCR Purification Kits (Thermo Fisher Scientific, Waltham MA, USA), Nucleospin Gel and PCR Clean-up (Machery-Nagel GmbH, Düren, Germany) or Monarch PCR and DNA Cleanup kits (NEB).

Techniques: Plasmid Preparation, Amplification, Polymerase Chain Reaction, Clone Assay

Journal: Nucleic Acids Research

Article Title: Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis

doi: 10.1093/nar/gky067

Figure Lengend Snippet: Darwin assembled TgoT DNA polymerase library. ( A ) Five separate sequencing reactions (range and reads shown in blue) were required to sample the diversity introduced across the eight target residues (shown in red along the TgoT gene). Mutations included focused degeneracies (e.g. YWC used against Y384) or ‘small intelligent’ (S-int) diversity (NDT, VMA, ATG and TGG oligonucleotides mixed in a 12:6:1:1 ratio). Resulting incorporation is shown in box plots with outliers explicitly labelled. Wild-type contamination was determined from positions where diversity excluded those sequences (N.A.: not applicable). As with the T7 RNA polymerase library, incorporation trends and biases were analysed to identify any biases in assembly. Ranked incorporation frequencies are shown for the residues targeted with ‘small intelligent’ diversity, and the top three highest (greens) and lowest (oranges) ranked codons (based on a straight sum of ranks) are highlighted ( B ). Outnest PCR of the TgoT DNA polymerase library (expected product of 2501 bp) showing that the final PCR can be carried out with either A- (MyTaq) or B-family (Q5) polymerases ( C ). MW: 1 kb ladder (NEB). NT: no template PCR control.

Article Snippet: PCR products were purified using GeneJET PCR Purification Kits (Thermo Fisher Scientific, Waltham MA, USA), Nucleospin Gel and PCR Clean-up (Machery-Nagel GmbH, Düren, Germany) or Monarch PCR and DNA Cleanup kits (NEB).

Techniques: Sequencing, Polymerase Chain Reaction

Journal: Nucleic Acids Research

Article Title: Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis

doi: 10.1093/nar/gky067

Figure Lengend Snippet: Principles of Darwin Assembly. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the cut strand degraded by exonuclease III (1). Boundary and inner (mutagenic) oligonucleotides are annealed to the ssDNA plasmid (2). Key features of the oligonucleotides are highlighted: 5′-boundary oligonucleotide is 5′-biotinylated; non-complementary overhangs are shown in blue with Type IIs endonuclease recognition sites shown in white; mutations are shown as red X in the inner oligonucleotides; 3′-boundary oligonucleotide is protected at its 3′-end. After annealing, primers are extended and ligated in an isothermal assembly reaction (3). The assembled strand can be isolated by paramagnetic streptavidin-coated beads (4) and purified by alkali washing prior to PCR using outnested priming sites (5) and cloning (6) using the type IIS restriction sites (white dots). The purification step (4) is not always necessary but we found it improved PCR performance, especially for long assembly reactions ( > 1 kb).

Article Snippet: PCR products were purified using GeneJET PCR Purification Kits (Thermo Fisher Scientific, Waltham MA, USA), Nucleospin Gel and PCR Clean-up (Machery-Nagel GmbH, Düren, Germany) or Monarch PCR and DNA Cleanup kits (NEB).

Techniques: Plasmid Preparation, Isolation, Purification, Polymerase Chain Reaction, Clone Assay

Journal: Frontiers in Pharmacology

Article Title: Aging-Associated Differences in Epitranscriptomic m6A Regulation in Response to Acute Cardiac Ischemia/Reperfusion Injury in Female Mice

doi: 10.3389/fphar.2021.654316

Figure Lengend Snippet: In vitro hypoxia/reperfusion (iH/R) stress regulates m6A levels of key anti-apoptotic and pro-apoptotic genes. ( A–C ). Ctrl and Mettl3 mofified HL1 cardiomyocytes were subjected to sham or iH/R, and total RNA was subjected to m6A RIP, followed by RT-qPCR using primers for the indicated pro-apoptotic genes (Bax and PTEN) and anti-apoptitic gene (Bcl2). Values are normalized to input. n = 3–5, * p

Article Snippet: Briefly, 2 µl N6-Methyladenosine Antibody was added to protein G magnetic beads (Thermo Fisher) and incubated at 4°C for 2 h. Following two washes in reaction buffer, the RNA was added to the antibody–bead mixture containing RNasin Plus RNase Inhibitor (Promega) and incubated at 4°C for 2 h. Next, RIP-enriched RNAs were isolated from the antibody-immobilized protein G beads using Monarch RNA cleanup binding buffer from Monarch RNA Cleanup kit (New England Biolabs).

Techniques: In Vitro, Quantitative RT-PCR