r0180  (New England Biolabs)

Journal: The Crispr Journal

Article Title: Transgenerational CRISPR-Cas9 Activity Facilitates Multiplex Gene Editing in Allopolyploid Wheat

doi: 10.1089/crispr.2017.0010

Figure Lengend Snippet: Transgenerational CRISPR-Cas9 activity induces new mutations in the TaGW2 and TaLpx-1 genes. NGS reads flanking the GW2T2 target site and their frequencies in (A) T 0 line GLM-2, (B) T 1 line GLM-2-9, and (C) T 2 line GLM-2-9-49 are shown. (D) Restriction enzyme digestion of polymerase chain reaction (PCR) amplicons to screen gw2 knockout mutations in the T 3 progenies of line GLM-2-9-49. The GW2T2 flanking region was amplified by PCR and digested with XmaI; non-digested PCR amplicons correspond to mutated GW2T2 target sites. The numbers on the gel image are identifiers of the GLM-2-9-49 progenies. Lanes marked with arrows are PCR products from wild-type plant not digested with XmaI and loaded as controls; the knockout mutant plant was marked with a star. BW, wild-type cultivar Bobwhite. (E) Sanger sequencing of PCR-amplified GW2T2 target sites of T 3 line GLM-2-9-49-28. Genome specific primers were used to amplify regions flanking the GW2T2 target sites. Nucleotide substitutions are marked with red rectangles, and the inserted nucleotide is shown by the red arrow. Types and frequencies of mutations at the GW2T2, LPX1T2, and MLOT1 target sites in (F) T 1 line GLM-2-5, and (G) T 2 line GLM-2-5-24 are shown. WT, wild-type alleles in wheat cultivar Bobwhite; “–” and “+” signs and numbers after them, nucleotides deleted and inserted, respectively. The frequency of each mutation type is shown on the right. The PAM sequences are underlined; the deleted nucleotides are shown with red dashed lines; the insertions and deletions are highlighted in red.

Article Snippet: To screen the gw2 knockout mutants, the GW2T2 target region from all three homoeologs was amplified, and PCR products were digested with XmaI (NEB).

Techniques: CRISPR, Activity Assay, Next-Generation Sequencing, Polymerase Chain Reaction, Knock-Out, Amplification, Mutagenesis, Sequencing

Journal: The Crispr Journal

Article Title: Transgenerational CRISPR-Cas9 Activity Facilitates Multiplex Gene Editing in Allopolyploid Wheat

doi: 10.1089/crispr.2017.0010

Figure Lengend Snippet: Transgenerational CRISPR-Cas9 activity induces new mutations in the TaGW2 and TaLpx-1 genes. NGS reads flanking the GW2T2 target site and their frequencies in (A) T 0 line GLM-2, (B) T 1 line GLM-2-9, and (C) T 2 line GLM-2-9-49 are shown. (D) Restriction enzyme digestion of polymerase chain reaction (PCR) amplicons to screen gw2 knockout mutations in the T 3 progenies of line GLM-2-9-49. The GW2T2 flanking region was amplified by PCR and digested with XmaI; non-digested PCR amplicons correspond to mutated GW2T2 target sites. The numbers on the gel image are identifiers of the GLM-2-9-49 progenies. Lanes marked with arrows are PCR products from wild-type plant not digested with XmaI and loaded as controls; the knockout mutant plant was marked with a star. BW, wild-type cultivar Bobwhite. (E) Sanger sequencing of PCR-amplified GW2T2 target sites of T 3 line GLM-2-9-49-28. Genome specific primers were used to amplify regions flanking the GW2T2 target sites. Nucleotide substitutions are marked with red rectangles, and the inserted nucleotide is shown by the red arrow. Types and frequencies of mutations at the GW2T2, LPX1T2, and MLOT1 target sites in (F) T 1 line GLM-2-5, and (G) T 2 line GLM-2-5-24 are shown. WT, wild-type alleles in wheat cultivar Bobwhite; “–” and “+” signs and numbers after them, nucleotides deleted and inserted, respectively. The frequency of each mutation type is shown on the right. The PAM sequences are underlined; the deleted nucleotides are shown with red dashed lines; the insertions and deletions are highlighted in red.

Article Snippet: Screening of gw2 knockout mutants To screen the gw2 knockout mutants, the GW2T2 target region from all three homoeologs was amplified, and PCR products were digested with XmaI (NEB).

Techniques: CRISPR, Activity Assay, Next-Generation Sequencing, Polymerase Chain Reaction, Knock-Out, Amplification, Mutagenesis, Sequencing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Gene delivery: A single nuclear localization signal peptide is sufficient to carry DNA to the cell nucleus

doi:

Figure Lengend Snippet: Strategy for the preparation of a double-stranded DNA fragment coupled to an NLS peptide. A functional luciferase gene of 3,380 bp was cut out of pCMVLuc with Xma I and Sal I. Further digestion with Xmn I and Bsp HI cut the unwanted restriction fragment into small fragments (970, 875, 768, and 240 bp) that were removed by sucrose gradient centrifugation. The capped CMVLuc-NLS DNA was obtained by ligation of the 32 P-labeled (∗) oligonucleotide-peptide and oligonucleotide-cap hairpins to the restriction fragment.

Article Snippet: Xma I, Xmn I, Sal I, and Bsp HI restriction endonucleases, T4 polynucleotide kinase, T4 DNA ligase, and exonuclease III were purchased from New England Biolabs.

Techniques: Functional Assay, Luciferase, Gradient Centrifugation, Ligation, Labeling

Journal: Blood Advances

Article Title: Simple and robust methylation test for risk stratification of patients with juvenile myelomonocytic leukemia

doi: 10.1182/bloodadvances.2021005080

Figure Lengend Snippet: Performance evaluation of DREAM. (A) Schematic outline of the principles of DNA methylation analysis. In DREAM, DNA is sequentially cut using 2 restriction enzymes, Sma I and Xma I. Estimated durations are indicated alongside each step. Individual sample sequencing could be completed in 4 to 5 days, including Sma I digestion (3 hours), Xma I digestion (16 hours), end repair (2 hours), adaptor ligation (1 hour), PCR amplification (2 hours), final library quality control (1 day), sequencing (2-3 days), and data analysis time. The hands-on time was

Article Snippet: Thereafter, 12.5 units of Xma I (New England Biolabs) was added, and digestion was continued for an additional 16 hours at 37°C.

Techniques: DNA Methylation Assay, Sequencing, Ligation, Polymerase Chain Reaction, Amplification