spei hf  (New England Biolabs)


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    Structured Review

    New England Biolabs spei hf
    Complexity Reduction Via Restriction Enzyme Degradation Increases the Number of On-Target Reads Number of CCS reads (individual molecules) is plotted for each of the four target regions ( C9orf72 , HTT , FMR1 , and ATXN10 ). Colors represent the amount of complexity reduction employed (blue = none; orange = two restriction enzymes (KpnI-HF and <t>MfeI-HF),</t> green = four restriction enzymes (KpnI-HF, Mfe-HFI, <t>SpeI-HF,</t> EcoRV-HF)).
    Spei Hf, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/spei hf/product/New England Biolabs
    Average 95 stars, based on 39 article reviews
    Price from $9.99 to $1999.99
    spei hf - by Bioz Stars, 2022-08
    95/100 stars

    Images

    1) Product Images from "Amplification-free, CRISPR-Cas9 Targeted Enrichment and SMRT Sequencing of Repeat-Expansion Disease Causative Genomic Regions"

    Article Title: Amplification-free, CRISPR-Cas9 Targeted Enrichment and SMRT Sequencing of Repeat-Expansion Disease Causative Genomic Regions

    Journal: bioRxiv

    doi: 10.1101/203919

    Complexity Reduction Via Restriction Enzyme Degradation Increases the Number of On-Target Reads Number of CCS reads (individual molecules) is plotted for each of the four target regions ( C9orf72 , HTT , FMR1 , and ATXN10 ). Colors represent the amount of complexity reduction employed (blue = none; orange = two restriction enzymes (KpnI-HF and MfeI-HF), green = four restriction enzymes (KpnI-HF, Mfe-HFI, SpeI-HF, EcoRV-HF)).
    Figure Legend Snippet: Complexity Reduction Via Restriction Enzyme Degradation Increases the Number of On-Target Reads Number of CCS reads (individual molecules) is plotted for each of the four target regions ( C9orf72 , HTT , FMR1 , and ATXN10 ). Colors represent the amount of complexity reduction employed (blue = none; orange = two restriction enzymes (KpnI-HF and MfeI-HF), green = four restriction enzymes (KpnI-HF, Mfe-HFI, SpeI-HF, EcoRV-HF)).

    Techniques Used:

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    New England Biolabs spei hf
    Complexity Reduction Via Restriction Enzyme Degradation Increases the Number of On-Target Reads Number of CCS reads (individual molecules) is plotted for each of the four target regions ( C9orf72 , HTT , FMR1 , and ATXN10 ). Colors represent the amount of complexity reduction employed (blue = none; orange = two restriction enzymes (KpnI-HF and <t>MfeI-HF),</t> green = four restriction enzymes (KpnI-HF, Mfe-HFI, <t>SpeI-HF,</t> EcoRV-HF)).
    Spei Hf, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/spei hf/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    spei hf - by Bioz Stars, 2022-08
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      Buy from Supplier

    96
    New England Biolabs spei ecorv hf
    Isolation of genetically corrected recessive dystrophic epidermolysis bullosa (RDEB) epidermal stem cells Single cells were isolated from a mass culture (passage V) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA. Clonal types were determined (Barrandon Green, 1987 ) and listed in Supplementary Table S1. Growing clones were expanded for further characterisation. COLVII detection in clones by immunostaining. COLVII expression (green) was detectable in some clones (6, 17, 22, 58 and 61) and not in others (3, 24 and 54); nuclei were stained with Hoechst 33342 (blue). Dotted lines delimit the periphery of keratinocyte colonies from the surrounding irradiated 3T3-J2 feeder cells. Scale bar: 50 μm. Quantitative RT–PCR analysis of COL7A1 expression in transduced clones compared to untransduced RDEB keratinocytes. All clones shown in (A) were transduced but expressed different levels of COL7A1 transcripts. Clones 6, 17, 22, 54, 58 and 61 expressed higher levels of COL7A1 than control RDEB cells and keratinocytes obtained from healthy donors (YF29 and OR-CA, control 1 and 2, respectively). The level of COL7A1 expression in the RDEB untransduced cells was referenced as 1. Determination of proviral rearrangements in transduced clones. A Southern blot was performed using genomic DNA of RDEB cells, clones and the infected mass culture from which the clones were isolated. Genomic DNA was digested with <t>EcoRV</t> and <t>SpeI</t> that cut at the 3′ and 5′ end of the provirus (Supplementary Fig S2) and hybridised with a 907-bp COL7A1 probe radiolabelled with 32 P isotope. The upper band corresponded to the endogenous signal. The retroviral producer line Flp293A-E1aColVII1 was used as a control for the digested 9.6-kb provirus (proviral signal). Smaller bands corresponded to rearranged proviruses marked with an asterisk. Identification of stem cells producing COLVII. Western blotting revealed that only clone 6 secreted COLVII in the culture supernatant, while clone 54 and surprisingly clone 22 did not (see A). RDEB cells were used as a negative control and healthy donor cells as a positive control. The secreted matrix metalloproteinase 2 (MMP2) was used as a loading control.
    Spei Ecorv Hf, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Complexity Reduction Via Restriction Enzyme Degradation Increases the Number of On-Target Reads Number of CCS reads (individual molecules) is plotted for each of the four target regions ( C9orf72 , HTT , FMR1 , and ATXN10 ). Colors represent the amount of complexity reduction employed (blue = none; orange = two restriction enzymes (KpnI-HF and MfeI-HF), green = four restriction enzymes (KpnI-HF, Mfe-HFI, SpeI-HF, EcoRV-HF)).

    Journal: bioRxiv

    Article Title: Amplification-free, CRISPR-Cas9 Targeted Enrichment and SMRT Sequencing of Repeat-Expansion Disease Causative Genomic Regions

    doi: 10.1101/203919

    Figure Lengend Snippet: Complexity Reduction Via Restriction Enzyme Degradation Increases the Number of On-Target Reads Number of CCS reads (individual molecules) is plotted for each of the four target regions ( C9orf72 , HTT , FMR1 , and ATXN10 ). Colors represent the amount of complexity reduction employed (blue = none; orange = two restriction enzymes (KpnI-HF and MfeI-HF), green = four restriction enzymes (KpnI-HF, Mfe-HFI, SpeI-HF, EcoRV-HF)).

    Article Snippet: To improve the observed on-target capture rate, a genome complexity reduction step was added before library preparation by pre-digesting genomic DNA samples with high fidelity restriction enzymes, KpnI-HF, MfeI-HF, SpeI-HF, and EcoRV-HF (New England Biolabs), in the presence of calf intestinal alkaline phosphatase (CIP, New England Biolabs).

    Techniques:

    Schematic representation of the QGA protocol. ( a ) Preparation of a DNA fragment containing a BioBrick part. P: prefix DNA fragment, BB: BioBrick, S: suffix DNA fragment, 100 bp UP-F: 100 base pair upstream forward primer, 200 bp DN-R: 200 base pair downstream reverse primer, circled P: phosphate residue, X: Xba I restriction site. ( b ) Structural change of extending the DNA molecule in each step of QGA. MAGB: magnetic beads, E: Eco RI restriction site, S: Spe I restriction site, S/X: mixed restriction site (scar), TTT…TTT: oligo-dT conjugated to magnetic beads, AAA…AAA: oligo-dA in DNA adaptor.

    Journal: Synthetic Biology

    Article Title: BioBrick-based ‘Quick Gene Assembly’ in vitro

    doi: 10.1093/synbio/ysx003

    Figure Lengend Snippet: Schematic representation of the QGA protocol. ( a ) Preparation of a DNA fragment containing a BioBrick part. P: prefix DNA fragment, BB: BioBrick, S: suffix DNA fragment, 100 bp UP-F: 100 base pair upstream forward primer, 200 bp DN-R: 200 base pair downstream reverse primer, circled P: phosphate residue, X: Xba I restriction site. ( b ) Structural change of extending the DNA molecule in each step of QGA. MAGB: magnetic beads, E: Eco RI restriction site, S: Spe I restriction site, S/X: mixed restriction site (scar), TTT…TTT: oligo-dT conjugated to magnetic beads, AAA…AAA: oligo-dA in DNA adaptor.

    Article Snippet: Restriction endonucleases, Xba I (20 U/μl), Spe I-HF (20 U/μl), Spe I (10 U/μl), EcoRI (20 U/μl), and solution for digestion (CutSmart) were purchased from New England Biolabs Japan Inc (Tokyo, Japan).

    Techniques: Magnetic Beads

    Negative effects on the QGA process resulting from the change of each condition away from optimum conditions. Lane 1: assembled products under optimum conditions. Lane 2: replacement of the restriction enzyme from Spe I-HF (NEB) to Spe I (NEB). Lane 3: changing of ligation condition from ‘at 16 °C for 30 min’ to ‘at 25 °C for 10 min’. Lane 4: removal of 0.1% Triton X-100 from the rinsing solution. Lane 5: removal of 0.6 M trehalose from the digestion premix. Lane 6: combination of all changes tested from lanes 2–5. Copy numbers of PtetR fragment are indicated at the right side of each band.

    Journal: Synthetic Biology

    Article Title: BioBrick-based ‘Quick Gene Assembly’ in vitro

    doi: 10.1093/synbio/ysx003

    Figure Lengend Snippet: Negative effects on the QGA process resulting from the change of each condition away from optimum conditions. Lane 1: assembled products under optimum conditions. Lane 2: replacement of the restriction enzyme from Spe I-HF (NEB) to Spe I (NEB). Lane 3: changing of ligation condition from ‘at 16 °C for 30 min’ to ‘at 25 °C for 10 min’. Lane 4: removal of 0.1% Triton X-100 from the rinsing solution. Lane 5: removal of 0.6 M trehalose from the digestion premix. Lane 6: combination of all changes tested from lanes 2–5. Copy numbers of PtetR fragment are indicated at the right side of each band.

    Article Snippet: Restriction endonucleases, Xba I (20 U/μl), Spe I-HF (20 U/μl), Spe I (10 U/μl), EcoRI (20 U/μl), and solution for digestion (CutSmart) were purchased from New England Biolabs Japan Inc (Tokyo, Japan).

    Techniques: Ligation

    Isolation of genetically corrected recessive dystrophic epidermolysis bullosa (RDEB) epidermal stem cells Single cells were isolated from a mass culture (passage V) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA. Clonal types were determined (Barrandon Green, 1987 ) and listed in Supplementary Table S1. Growing clones were expanded for further characterisation. COLVII detection in clones by immunostaining. COLVII expression (green) was detectable in some clones (6, 17, 22, 58 and 61) and not in others (3, 24 and 54); nuclei were stained with Hoechst 33342 (blue). Dotted lines delimit the periphery of keratinocyte colonies from the surrounding irradiated 3T3-J2 feeder cells. Scale bar: 50 μm. Quantitative RT–PCR analysis of COL7A1 expression in transduced clones compared to untransduced RDEB keratinocytes. All clones shown in (A) were transduced but expressed different levels of COL7A1 transcripts. Clones 6, 17, 22, 54, 58 and 61 expressed higher levels of COL7A1 than control RDEB cells and keratinocytes obtained from healthy donors (YF29 and OR-CA, control 1 and 2, respectively). The level of COL7A1 expression in the RDEB untransduced cells was referenced as 1. Determination of proviral rearrangements in transduced clones. A Southern blot was performed using genomic DNA of RDEB cells, clones and the infected mass culture from which the clones were isolated. Genomic DNA was digested with EcoRV and SpeI that cut at the 3′ and 5′ end of the provirus (Supplementary Fig S2) and hybridised with a 907-bp COL7A1 probe radiolabelled with 32 P isotope. The upper band corresponded to the endogenous signal. The retroviral producer line Flp293A-E1aColVII1 was used as a control for the digested 9.6-kb provirus (proviral signal). Smaller bands corresponded to rearranged proviruses marked with an asterisk. Identification of stem cells producing COLVII. Western blotting revealed that only clone 6 secreted COLVII in the culture supernatant, while clone 54 and surprisingly clone 22 did not (see A). RDEB cells were used as a negative control and healthy donor cells as a positive control. The secreted matrix metalloproteinase 2 (MMP2) was used as a loading control.

    Journal: EMBO Molecular Medicine

    Article Title: A single epidermal stem cell strategy for safe ex vivo gene therapy

    doi: 10.15252/emmm.201404353

    Figure Lengend Snippet: Isolation of genetically corrected recessive dystrophic epidermolysis bullosa (RDEB) epidermal stem cells Single cells were isolated from a mass culture (passage V) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA. Clonal types were determined (Barrandon Green, 1987 ) and listed in Supplementary Table S1. Growing clones were expanded for further characterisation. COLVII detection in clones by immunostaining. COLVII expression (green) was detectable in some clones (6, 17, 22, 58 and 61) and not in others (3, 24 and 54); nuclei were stained with Hoechst 33342 (blue). Dotted lines delimit the periphery of keratinocyte colonies from the surrounding irradiated 3T3-J2 feeder cells. Scale bar: 50 μm. Quantitative RT–PCR analysis of COL7A1 expression in transduced clones compared to untransduced RDEB keratinocytes. All clones shown in (A) were transduced but expressed different levels of COL7A1 transcripts. Clones 6, 17, 22, 54, 58 and 61 expressed higher levels of COL7A1 than control RDEB cells and keratinocytes obtained from healthy donors (YF29 and OR-CA, control 1 and 2, respectively). The level of COL7A1 expression in the RDEB untransduced cells was referenced as 1. Determination of proviral rearrangements in transduced clones. A Southern blot was performed using genomic DNA of RDEB cells, clones and the infected mass culture from which the clones were isolated. Genomic DNA was digested with EcoRV and SpeI that cut at the 3′ and 5′ end of the provirus (Supplementary Fig S2) and hybridised with a 907-bp COL7A1 probe radiolabelled with 32 P isotope. The upper band corresponded to the endogenous signal. The retroviral producer line Flp293A-E1aColVII1 was used as a control for the digested 9.6-kb provirus (proviral signal). Smaller bands corresponded to rearranged proviruses marked with an asterisk. Identification of stem cells producing COLVII. Western blotting revealed that only clone 6 secreted COLVII in the culture supernatant, while clone 54 and surprisingly clone 22 did not (see A). RDEB cells were used as a negative control and healthy donor cells as a positive control. The secreted matrix metalloproteinase 2 (MMP2) was used as a loading control.

    Article Snippet: Ten micrograms of DNA was codigested with SpeI/EcoRV HF (New England Biolabs) and loaded on a 0.8% agarose (Promega) gel.

    Techniques: Isolation, Infection, Clone Assay, Immunostaining, Expressing, Staining, Irradiation, Quantitative RT-PCR, Southern Blot, Western Blot, Negative Control, Positive Control