monarch gdna blood lysis buffer  (New England Biolabs)

Journal: bioRxiv

Article Title: Efficient genome editing in multiple salmonid cell lines using ribonucleoprotein complexes

doi: 10.1101/2020.04.03.022038

Figure Lengend Snippet: SHK-1 cells were electroporated with 1.4 μM Cas9:gRNA RNP and transferred to 2 separate 96-well plates. (A) After 48h, cell survival (plate1) was calculated using CellTiter Glo 2.0. (B) genomic DNA (plate 2) was extracted at 7 dpt, and the target sequence amplified by PCR and editing efficiency estimated using Sanger sequencing. (C) Using 1.4 μM RNP, the editing efficiency after 7 and 14 ddpt was estimated for different electroporation settings. (D) All the sequencing data, obtained from ICE analysis of Sanger sequencing of the intergenic target region from optimisation experiments (n=55) were pooled and plotted according to edit pattern.

Article Snippet: Genomic DNA (gDNA) was extracted with QuickExtract buffer (Lucigen, Middleton, USA) by adding 30 μL to a well of a 96-well plate and incubating for 5 min.

Techniques: Sequencing, Amplification, Polymerase Chain Reaction, Electroporation

Journal: Cell

Article Title: SAF-A Regulates Interphase Chromosome Structure through Oligomerization with Chromatin-Associated RNAs

doi: 10.1016/j.cell.2017.05.029

Figure Lengend Snippet: SAF-A Oligomerization Regulates Interphase Chromatin Structure via Chromatin-Associated RNAs but Is Not Required to Maintain C 0 t1 and LINE-1 RNA, Related to Figure 6 (A) Boxplots showing the distribution of distances between probe pairs at 2p25.1 from a FISH chromatin compaction assay in cells before (0 hr, red rectangle, box 1), after SAF-A depletion (siSAF-A, dark orange rectangle, box 2) and after re-expression of siRNA-resistant wild-type SAF-A, Walker A or Walker B mutants in the absence (pale orange rectangles, box 3, 5, 7) or presence (green rectangles, box 4, 6, 8) of α-amanitin (n > 100). Colored bars correspond to time points shown in Figure 6 A. (B) Immunofluorescence for SAF-A in Triton X-100 extracted RPE1 cells treated with RNaseA/T1, counterstained with DAPI. Scale bar, 10μm. (C) DNA-FISH for human chromosome 3 (HSA3, C 0 t1 DNA probe) in a human-hamster hybrid cell line (GM10253A) that stably carries HSA3 in conjunction with RNA-FISH to assay for the binding and distribution of caRNAs (C 0 t1 RNA probe) or LINE-1 ORF2 RNA in control cells (siControl) and cells depleted for SAF-A (siSAF-A) and cells treated with the transcription inhibitors α-amanitin or actinomycin D. Scale bars, 10 μm. (D) Boxplot showing area occupied by HSA3 territory (C 0 t1 DNA probe) as in panel C in GM10253A control cells (siControl), cells depleted for SAF-A (siSAF-A) and cells treated with the transcription inhibitors α-amanitin or actinomycin D (5 h). P values for a Wilcoxon test (n > 30 nuclei; ∗∗∗∗ p

Article Snippet: The precipitate was suspended in 70% ethanol overnight and resuspended in 50 μL of combined DNA and RNA lysis buffer (1 × DNaseI digestion buffer (NEB) with 1 mM ZnSO4).

Techniques: Fluorescence In Situ Hybridization, Expressing, Immunofluorescence, Stable Transfection, Binding Assay

Journal: Cell

Article Title: SAF-A Regulates Interphase Chromosome Structure through Oligomerization with Chromatin-Associated RNAs

doi: 10.1016/j.cell.2017.05.029

Figure Lengend Snippet: Structural Characteristics of SAF-A and Identification of Walker and Initiator-Specific Motifs, Related to Figure 3 (A) SAF-A encodes four conserved domains: SAP (SAF-A/B, Acinus and PIAS) ( Aravind and Koonin, 2000 ) which has DNA binding activity ( Göhring et al., 1997 ), SPRY (Spla and Ryanodine receptor) of unknown function ( Ponting et al., 1997 ), AAA+ (ATPases Associated with diverse cellular Activities) ( Erzberger and Berger, 2006 ), and a low complexity RGG (arginine glycine-glycine) RNA-binding domain ( Helbig and Fackelmayer, 2003 , Thandapani et al., 2013 ). Probability of protein disorder across SAF-A calculated using different algorithms. Amino acid number is given on x axis while y axis depicts the levels of protein disorder. (B) Sequence and structure homology between SAF-A AAA+ domain and mammalian PNK (PDB-3ZVL) structure showing predicted α helices, β sheets and putative Walker motifs. Conserved amino acids are labeled in red and similar amino acids are marked in yellow. (C) Left, predicted ribbon diagram of SAF-A, modeled on PDB-3ZVL, showing putative Walker motifs and ISM. Right, cartoon of SAF-A protein showing key domains and ISM.

Article Snippet: The precipitate was suspended in 70% ethanol overnight and resuspended in 50 μL of combined DNA and RNA lysis buffer (1 × DNaseI digestion buffer (NEB) with 1 mM ZnSO4).

Techniques: Binding Assay, Activity Assay, RNA Binding Assay, Sequencing, Labeling

Journal: Scientific Reports

Article Title: Efficient genome editing of an extreme thermophile, Thermus thermophilus, using a thermostable Cas9 variant

doi: 10.1038/s41598-021-89029-2

Figure Lengend Snippet: CaldoCas9 based genome editing of T. thermophilus at 65 °C. ( A ) pTTCC_crtI transformants streaked out on fresh media and grown overnight to reveal pigmentation difference in the transformants and WT cells. ( B ) Agarose gel electrophoresis of products from colony PCR amplification of the genomic locus containing crtI in T. thermophilus pTTCC_crtI transformants. Expected band size of the KO allele was 1351 bp and of WT allele 2891 bp. ( C ) pTTCC_purA transformants streaked out on minimal media with and without adenine supplementation (+ Ade − Ade, respectively) to reveal the adenine auxotrophic phenotype. ( D ) Agarose gel electrophoresis of products from colony PCR amplification of genomic locus containing purA in T. thermophilus pTTCC_purA transformants. Expected band size of the KO allele was 1094 bp and the WT allele 2320 bp. ( E ) After growth of pTTCC_purA and pTTCC_crtI transformants in media without antibiotic selection, individual colonies were streaked out on media with and without kanamycin (+ Kan, − Kan, respectively) to reveal the presence or absence of the plasmid. The agarose gel images ( B , D ) have been digitally manipulated for clarity. The original images are provided in supplementary file 5 .

Article Snippet: A few clones were cultured overnight in liquid L media with kanamycin (30 µg/ml) and plasmids miniprepped (NEB, T1010).

Techniques: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Selection, Plasmid Preparation

Journal: Microorganisms

Article Title: EpicPCR 2.0: Technical and Methodological Improvement of a Cutting-Edge Single-Cell Genomic Approach

doi: 10.3390/microorganisms9081649

Figure Lengend Snippet: Efficiencies of two- and three-steps epicPCR protocols using different blocking primers (BPs) concentrations. The epicPCRs were run on SXT/R391 carrying bacteria from the Meurthe River water. A and C indicate wells with amplification products from two-steps epicPCR protocols (fusion-PCR on polyacrylamide beads + nested PCR). In the A lane, fusion-PCR products from the first step were used as template DNA in the second step without dilution whereas these products were diluted ten times in line C to circumvent the possible presence of PCR inhibitors. B indicates wells loaded with amplification products resulting from a three-steps epicPCR protocol (fusion PCR on polyacrylamide beads + blocking PCR with BPs as sole primers + nested PCR). The expected size of the final nested-PCR product is around 350 bp (depending on the 16S rRNA gene fragment polymorphism). The conditions of epicPCR as used in Hultman et al. (2018) and that we determined to be the best in our conditions (epicPCR 2.0) are indicated by black arrows. The minus symbols indicate negative controls with epicPCRs run without polyacrylamide beads-template.

Article Snippet: The PCR products were pooled, cleaned (Monarch PCR & DNA Cleanup Kit; New England Biolabs, Ipswich, MA, USA) and sequenced on Illumina Miseq (2 × 250) (Genewiz, South Plainfield, NJ, USA).

Techniques: Blocking Assay, Amplification, Polymerase Chain Reaction, Nested PCR

Journal: Microorganisms

Article Title: EpicPCR 2.0: Technical and Methodological Improvement of a Cutting-Edge Single-Cell Genomic Approach

doi: 10.3390/microorganisms9081649

Figure Lengend Snippet: Control epicPCR amplifications targeting SXT/R391 ICEs performed on beads carrying E. coli MG1656::SXT MO10 as template. Each experiment was done in duplicates with a no-template DNA control, using either the Phusion DNA Polymerase GC or HF buffers. In the nested-PCR step, the use of blocking primers (BPs) has been done as depicted in Spencer et al. (2016), and usually performed so far. All these conditions are summarized in the table upper the gel. The expected size of the desired DNA fragment is 367 bp. Black arrows indicate epicPCR products obtained after performing fusion and nested PCRs using HF but not GC buffer.

Article Snippet: The PCR products were pooled, cleaned (Monarch PCR & DNA Cleanup Kit; New England Biolabs, Ipswich, MA, USA) and sequenced on Illumina Miseq (2 × 250) (Genewiz, South Plainfield, NJ, USA).

Techniques: Nested PCR, Blocking Assay