sau96i  (New England Biolabs)

Journal: Revista do Instituto de Medicina Tropical de São Paulo

Article Title: The epidemiology of cryptococcosis and the characterization of Cryptococcus neoformans isolated in a Brazilian University Hospital

doi: 10.1590/S1678-9946201759013

Figure Lengend Snippet: RFLP profile of the URA5 genes from Cryptococcus sp obtained by double-digestion with HhaI and Sau96I. Column 1: Molecular maker (100 bp). Columns 2 to 7: isolated 2, 4, 5, 6, 7 and 8 (VNI), respectively; column 8: isolated 12 (VGI); Columns 9 to 13: isolates 13, 18, 22, 24 and 25 (VNI), respectively.

Article Snippet: Thirty microliters of the reaction amplicon were doubly digested with Sau96I (10 U/µL) and HhaI (20 U/µL) (New England Biolabs, Uniscience, SP, Brazil), and incubated at 37 °C for 3 hours.

Techniques: Isolation

Journal: PLoS ONE

Article Title: Challenges associated with homologous directed repair using CRISPR-Cas9 and TALEN to edit the DMD genetic mutation in canine Duchenne muscular dystrophy

doi: 10.1371/journal.pone.0228072

Figure Lengend Snippet: DNA and RNA analysis revealed HDR-mediated gene editing in GRMD-treated (Tx) myoblasts. (a) Agarose gel after PCR and restriction digest of GRMD-Tx and non-Tx cells. From left to right: Normal (N), carrier (Ca), non-Tx GRMD (md), GRMD-sgRNA A-Tx (A), GRMD-sgRNA B-Tx (B), GRMD-sgRNA A B combined Tx (A B), GRMD-TALEN Tx (T), ladder (100bp). All bands were sequenced: top band of ~ 700bp (red asterisk) matched part of the sequence of the donor clone after being cut with Sau96I enzyme, second band of ~ 500bp (red cross) was the corrected DMD gene sequence in GRMD-HDR-Tx samples and normal dog cells. This band was not cut with Sau96I. This second band had a higher molecular weight in GRMD-Tx cells compared to normal due to additional genes (eGFP) present in the donor clone. Third and fourth bands ~ 200bp (red dash, red cash sign) correspond to fragments of the GRMD mutated dog genome that was cut with Sau96I. (b) Sanger sequencing from the cut band (red cross) in a normal dog. Red arrow denotes the correct bp (A) in the DMD gene. (c) Sanger sequencing from the cut band in GRMD-HDR-Tx myoblasts but not successfully edited. Red arrow points at mutated bp (G) in GRMD dogs (d) Sanger sequencing from cut band in successfully GRMD-HDR-Tx GRMD cells. Red arrow denotes successfully replaced bp (A). (e) Dystrophin mRNA expression (mean±SE) among HDR treatments and normal cells compared to normal cells expression. *** p ≤ 0.001, ** p ≤ 0.01 vs Normal. Samples were analyzed using a pair wise fixed reallocation randomization test, excluding outliers with a Grubb’s test. Vertical bars indicate standard error of the mean. Treated myoblasts were differentiated into myotubes for 18 to 21 days and RNA was extracted from 6 replicates; values were normalized to HPRT1 (house-keeping gene).

Article Snippet: Sanger sequencing For DNA genotyping of cells, PCR bands that were restriction digested by Sau96I (New England Biolabs) were cut, ligated (T.A.

Techniques: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Sequencing, Molecular Weight, Expressing

Journal: PLoS ONE

Article Title: Challenges associated with homologous directed repair using CRISPR-Cas9 and TALEN to edit the DMD genetic mutation in canine Duchenne muscular dystrophy

doi: 10.1371/journal.pone.0228072

Figure Lengend Snippet: Experimental design of HDR-mediated CRISPR/Cas9 and TALEN gene editing for the GRMD mutation. (a) Guide selection included sgRNA A (PAM A underlined) and sgRNA B (PAM B underlined). (b) Experimental design. Double stranded breaks (DSB) occurred at the intron 6 area (highlighted) and/or at the exon 7 area to excise the GRMD mutation (asterisk). The donor clone (green) was used as a template for HDR to replace the excised area with the corrected DMD gene sequence. The black arrow designates the cutting site for Sau96I restriction enzyme, used to genotype GRMD dogs. When the dog does not have the mutated bp, Sau96I does not cut the DNA. (c) TALEN arm design with left and right sequences.

Article Snippet: Sanger sequencing For DNA genotyping of cells, PCR bands that were restriction digested by Sau96I (New England Biolabs) were cut, ligated (T.A.

Techniques: CRISPR, Mutagenesis, Selection, Sequencing

Journal: PLoS ONE

Article Title: A PCR plus restriction enzyme-based technique for detecting target-enzyme mutations at position Pro-106 in glyphosate-resistant Lolium perenne

doi: 10.1371/journal.pone.0246028

Figure Lengend Snippet: Digestion patterns of three restriction enzymes, Msc I (A), Sau 96I (B) and Nla IV (C) used for the dCAPS markers for detecting mutations at position Pro-106 in EPSPS gene in glyphosate-resistant population O (R) and glyphosate-susceptible population SP (S). S 1 and R 1 represent undigested samples showing the 216 bp dCAPS amplicon from an individual each of population S and O, respectively. S 2 represents restriction enzyme-digested samples for the glyphosate susceptible individual yielding a single ~180 bp fragment. This individual was homozygous for the wild-type glyphosate sensitive allele of the EPSPS gene that harbors the C nucleotide at the codon 106 required for restriction enzyme cleavage in these dCAPS assays. The glyphosate-resistant individual (R 2 ) had a 216 bp and ~180 bp fragment showing the presence of a mutation at codon 106 of the EPSPS gene for one allele as well as the wild-type sensitive allele, indicating this plant was heterozygous for these alleles. M denotes the DNA size standard lanes showing the 200 and 300 bp fragments of the 1 kb Plus size marker ladder (NEB, UK). The samples were resolved and visualized by electrophoresis in an agarose lithium borate buffer (2% w v -1 ) gel containing 0.5 μg ml -1 ethidium bromide.

Article Snippet: The restriction digestion reaction (10 μl) contained 8.5 μl of PCR products, 1 μl of the appropriate buffer and 0.5 μl (5 units) of Msc I and Sau 96I enzymes (NEB, UK) for Gly-dCAPS-F1/ Gly-dCAPS-R1 and Gly-dCAPS-F2/ Gly-dCAPS-R2 primers, respectively.

Techniques: Amplification, Mutagenesis, Marker, Electrophoresis

Journal: PLoS ONE

Article Title: A PCR plus restriction enzyme-based technique for detecting target-enzyme mutations at position Pro-106 in glyphosate-resistant Lolium perenne

doi: 10.1371/journal.pone.0246028

Figure Lengend Snippet: Digestion patterns of the restriction enzyme, Sau 96I used for the dCAPS markers for detecting mutations at codon 106 in the EPSPS gene in 18 individuals from a glyphosate-resistant Blenheim population. The presence of the 216 bp dCAPS amplicon indicated a non-cleaved resistant allele of the EPSPS gene, whereas the ~180 bp cleaved amplicon showed presence of the glyphosate-sensitive allele. RS and RR represent digested samples for glyphosate-resistant individuals that were heterozygous and homozygous, for SNP mutations conferring resistance at codon 106 in the EPSPS gene. M denotes the DNA size standard lanes showing the 200 and 300 bp fragments of the 1 kb Plus size marker ladder (NEB, UK). The samples were resolved and visualized by electrophoresis in an agarose lithium borate buffer (2% w v -1 ) gel containing 0.5 μg ml -1 ethidium bromide.

Article Snippet: The restriction digestion reaction (10 μl) contained 8.5 μl of PCR products, 1 μl of the appropriate buffer and 0.5 μl (5 units) of Msc I and Sau 96I enzymes (NEB, UK) for Gly-dCAPS-F1/ Gly-dCAPS-R1 and Gly-dCAPS-F2/ Gly-dCAPS-R2 primers, respectively.

Techniques: Amplification, Marker, Electrophoresis

Journal: Journal of Clinical Microbiology

Article Title: Genotyping Encephalitozoon cuniculi by Multilocus Analyses of Genes with Repetitive Sequences

doi: 10.1128/JCM.39.6.2248-2253.2001

Figure Lengend Snippet: Genotyping E. cuniculi isolates by PCR analysis of the PTP gene. (a) Differentiation of genotype III from genotypes I and II by eletrophoresis of PCR products: lanes 1 and 10, 100-bp ladders; lanes 2 (strain I), 4 (CDC:V385), 6 (CDC:V446), and 9 (CDC:V428A), genotype I; lane 8 (strain II), genotype II; and lanes 3 (CDC:V282), 5 (USP A-1), and 7 (3275), genotype III. (b) Differentiation of genotype I from genotype II by restriction digestion of PCR products with Sau 96I: lanes 2 (strain I), 3 (CDC:V385), 4 (CDC:V446), and 6 (CDC:V428A), genotype I; and lane 5 (strain II), genotype II.

Article Snippet: Although E. cuniculi genotypes I and II both generated PCR products of the same size, computer analysis of the PTP sequences obtained indicated that the 363-bp products of these two genotypes could be differentiated by the use of restriction enzymes Alw I, Mbo I, Bst YI, Dpn I, Fnu 4HI, Tse I, Bsa JI, Bbv I, Ava II, Ppu MI, or Sau 96I.

Techniques: Polymerase Chain Reaction