hindiii  (New England Biolabs)


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    Structured Review

    New England Biolabs hindiii
    Hindiii, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Isolation:

    Article Title: The Reduced Expression of 6ckine in the plt Mouse Results from the Deletion of One of Two 6ckine Genes
    Article Snippet: Tail DNA was isolated from 129/sv (The Jackson Laboratory) and BALB/c-plt mice. .. EcoRI and HindIII (New England Biolabs Inc.)–digested mouse tail DNA and BAC DNA were denatured and blotted onto Duralon membrane (Stratagene Inc.) and hybridized with probes A and B (see ).

    BAC Assay:

    Article Title: The Reduced Expression of 6ckine in the plt Mouse Results from the Deletion of One of Two 6ckine Genes
    Article Snippet: .. EcoRI and HindIII (New England Biolabs Inc.)–digested mouse tail DNA and BAC DNA were denatured and blotted onto Duralon membrane (Stratagene Inc.) and hybridized with probes A and B (see ). ..

    Southern Blot:

    Article Title: The Reduced Expression of 6ckine in the plt Mouse Results from the Deletion of One of Two 6ckine Genes
    Article Snippet: Paragraph title: Southern Blot Analysis of 6Ckine Locus. ... EcoRI and HindIII (New England Biolabs Inc.)–digested mouse tail DNA and BAC DNA were denatured and blotted onto Duralon membrane (Stratagene Inc.) and hybridized with probes A and B (see ).

    Mouse Assay:

    Article Title: The Reduced Expression of 6ckine in the plt Mouse Results from the Deletion of One of Two 6ckine Genes
    Article Snippet: Tail DNA was isolated from 129/sv (The Jackson Laboratory) and BALB/c-plt mice. .. EcoRI and HindIII (New England Biolabs Inc.)–digested mouse tail DNA and BAC DNA were denatured and blotted onto Duralon membrane (Stratagene Inc.) and hybridized with probes A and B (see ).

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    New England Biolabs hindiii digestion
    Visualization of nuclear compartments and chromatin domains in non-treated liver cells ( A ) and the same cells treated according to the 3C protocol up to the ligation step ( B ). The insoluble fraction was collected after <t>HindIII</t> digestion and 1.6% SDS extraction. (a–e) Immunostaining with antibodies against nucleolin (a), Sc35 (b), DNA topoisomerase II (c), H3K9me3 (d) and H3K27me3 (e). (f) Visualization of the chromosome 7 territory (FISH with a library of the chromosome 7–specific probes). In both sections of the Figure, the results of immunostaining are shown in the first row (red) and counterstaining of DNA with DAPI is shown in the second row (blue). The superimposition of the immunostaining and counterstaining of DNA is shown in the third row. Scale bar: 5 µm.
    Hindiii Digestion, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 80 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hindiii digestion/product/New England Biolabs
    Average 95 stars, based on 80 article reviews
    Price from $9.99 to $1999.99
    hindiii digestion - by Bioz Stars, 2020-02
    95/100 stars
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    Visualization of nuclear compartments and chromatin domains in non-treated liver cells ( A ) and the same cells treated according to the 3C protocol up to the ligation step ( B ). The insoluble fraction was collected after HindIII digestion and 1.6% SDS extraction. (a–e) Immunostaining with antibodies against nucleolin (a), Sc35 (b), DNA topoisomerase II (c), H3K9me3 (d) and H3K27me3 (e). (f) Visualization of the chromosome 7 territory (FISH with a library of the chromosome 7–specific probes). In both sections of the Figure, the results of immunostaining are shown in the first row (red) and counterstaining of DNA with DAPI is shown in the second row (blue). The superimposition of the immunostaining and counterstaining of DNA is shown in the third row. Scale bar: 5 µm.

    Journal: Nucleic Acids Research

    Article Title: Disclosure of a structural milieu for the proximity ligation reveals the elusive nature of an active chromatin hub

    doi: 10.1093/nar/gkt067

    Figure Lengend Snippet: Visualization of nuclear compartments and chromatin domains in non-treated liver cells ( A ) and the same cells treated according to the 3C protocol up to the ligation step ( B ). The insoluble fraction was collected after HindIII digestion and 1.6% SDS extraction. (a–e) Immunostaining with antibodies against nucleolin (a), Sc35 (b), DNA topoisomerase II (c), H3K9me3 (d) and H3K27me3 (e). (f) Visualization of the chromosome 7 territory (FISH with a library of the chromosome 7–specific probes). In both sections of the Figure, the results of immunostaining are shown in the first row (red) and counterstaining of DNA with DAPI is shown in the second row (blue). The superimposition of the immunostaining and counterstaining of DNA is shown in the third row. Scale bar: 5 µm.

    Article Snippet: The nuclei were harvested and suspended in 0.5 ml of 1.2× restriction buffer 2 (New England Biolabs) for subsequent HindIII digestion or 0.25 ml of 1.2× restriction buffer 3 (New England Biolabs) for MboI digestion.

    Techniques: Ligation, Immunostaining, Fluorescence In Situ Hybridization

    Electron microscopic analysis of the insoluble 3C material from liver cells at different steps of the 3C procedure. After formaldehyde cross-linking ( A and A’ ), after isolation of nuclei and extraction with 0.3% SDS followed by 1.8% Triton X-100 ( B and B’ ) and after digestion with HindIII restriction endonuclease followed by extraction with 1.6% SDS ( C and C’ ). Panels below show the enlarged framed region of the above images. Scale bars: 1 µm (A–C) and 250 nm (A’–C’).

    Journal: Nucleic Acids Research

    Article Title: Disclosure of a structural milieu for the proximity ligation reveals the elusive nature of an active chromatin hub

    doi: 10.1093/nar/gkt067

    Figure Lengend Snippet: Electron microscopic analysis of the insoluble 3C material from liver cells at different steps of the 3C procedure. After formaldehyde cross-linking ( A and A’ ), after isolation of nuclei and extraction with 0.3% SDS followed by 1.8% Triton X-100 ( B and B’ ) and after digestion with HindIII restriction endonuclease followed by extraction with 1.6% SDS ( C and C’ ). Panels below show the enlarged framed region of the above images. Scale bars: 1 µm (A–C) and 250 nm (A’–C’).

    Article Snippet: The nuclei were harvested and suspended in 0.5 ml of 1.2× restriction buffer 2 (New England Biolabs) for subsequent HindIII digestion or 0.25 ml of 1.2× restriction buffer 3 (New England Biolabs) for MboI digestion.

    Techniques: Isolation

    Frequencies of ligation of the fragment harboring the Hbb-b1 promoter with several selected fragments of the β-globin gene domain in soluble and insoluble portions of the 3C material. ( A ) Results of standard 3C analysis performed without fractionating the 3C material. ( B ) Results of 3C analysis performed separately on soluble (super) and insoluble (debris) fractions. ( C ) The same as (B) after normalization of the ligation frequencies to the amount of DNA in the samples. ( D ) The same as (C), soluble fraction only. On the top of each graph, a map of the domain is shown, with β-globin genes, olfactory receptor genes and DNase I hypersensitive sites shown by red arrows, blue arrows and black vertical lines, respectively. Plotted on the horizontal axis are the fragment positions. The scale is in kilobases, and according to GenBank entry NT_039433, the ‘0’ point corresponds to the start of the Hbb-y gene. The black rectangle in the background of each graph shows the anchor fragment, and the gray rectangles indicate test fragments. Plotted on the vertical axis are the ligation frequencies; the highest ligation frequency observed is set to 100 [the frequency of ligation between the anchor fragment and the upstream restriction fragment in the total 3C material from fetal liver cells (A) or in the insoluble portion of the 3C material from fetal liver cells (B and C) or the soluble portion of the 3C material from fetal brain cells (D)]. Red and blue lines show the results for liver and brain cells, respectively; solid lines show the results for the total 3C material (A) or the insoluble portion of the 3C material (B and C); dotted lines show the results for the soluble portion of the 3C material. Ligation frequencies of HindIII and MboI fragments are presented on the left and the right graphs, respectively. The error bars represent SEM for three independent experiments.

    Journal: Nucleic Acids Research

    Article Title: Disclosure of a structural milieu for the proximity ligation reveals the elusive nature of an active chromatin hub

    doi: 10.1093/nar/gkt067

    Figure Lengend Snippet: Frequencies of ligation of the fragment harboring the Hbb-b1 promoter with several selected fragments of the β-globin gene domain in soluble and insoluble portions of the 3C material. ( A ) Results of standard 3C analysis performed without fractionating the 3C material. ( B ) Results of 3C analysis performed separately on soluble (super) and insoluble (debris) fractions. ( C ) The same as (B) after normalization of the ligation frequencies to the amount of DNA in the samples. ( D ) The same as (C), soluble fraction only. On the top of each graph, a map of the domain is shown, with β-globin genes, olfactory receptor genes and DNase I hypersensitive sites shown by red arrows, blue arrows and black vertical lines, respectively. Plotted on the horizontal axis are the fragment positions. The scale is in kilobases, and according to GenBank entry NT_039433, the ‘0’ point corresponds to the start of the Hbb-y gene. The black rectangle in the background of each graph shows the anchor fragment, and the gray rectangles indicate test fragments. Plotted on the vertical axis are the ligation frequencies; the highest ligation frequency observed is set to 100 [the frequency of ligation between the anchor fragment and the upstream restriction fragment in the total 3C material from fetal liver cells (A) or in the insoluble portion of the 3C material from fetal liver cells (B and C) or the soluble portion of the 3C material from fetal brain cells (D)]. Red and blue lines show the results for liver and brain cells, respectively; solid lines show the results for the total 3C material (A) or the insoluble portion of the 3C material (B and C); dotted lines show the results for the soluble portion of the 3C material. Ligation frequencies of HindIII and MboI fragments are presented on the left and the right graphs, respectively. The error bars represent SEM for three independent experiments.

    Article Snippet: The nuclei were harvested and suspended in 0.5 ml of 1.2× restriction buffer 2 (New England Biolabs) for subsequent HindIII digestion or 0.25 ml of 1.2× restriction buffer 3 (New England Biolabs) for MboI digestion.

    Techniques: Ligation

    Southern blots of Hin dIII and Hin dIII-VDE digests of DNA from spo11 strains with inserts at HIS4 (top) and at URA3 (bottom). Gel labels are as in Figure 1 ; JM—joint molecule recombination intermediates. DOI: http://dx.doi.org/10.7554/eLife.19669.015

    Journal: eLife

    Article Title: Local chromosome context is a major determinant of crossover pathway biochemistry during budding yeast meiosis

    doi: 10.7554/eLife.19669

    Figure Lengend Snippet: Southern blots of Hin dIII and Hin dIII-VDE digests of DNA from spo11 strains with inserts at HIS4 (top) and at URA3 (bottom). Gel labels are as in Figure 1 ; JM—joint molecule recombination intermediates. DOI: http://dx.doi.org/10.7554/eLife.19669.015

    Article Snippet: Recombination products were detected on Southern blots containing genomic DNA digested with Hin dIII and VDE (P I-Sce I, New England Biolabs), using specific buffer for P I-Sce I.

    Techniques:

    Spo11-initiated events at the two insert loci. ( A ) Spo11-catalyzed DSBs are more frequent at HIS4 that at URA3 . Left—Southern blots of Eco RI digests of DNA from vde∆ strains, probed with pBR322 sequences, showing Spo11-DSBs in the Parent 2 insert (see Figure 1 ) in resection/repair-deficient sae2∆ mutant strains. Right—location of DSBs and probe and DSB frequencies (average of 7 and 8 hr samples from a single experiment; error bars represent range). Spo11-DSBs in the Parent 1 inserts at HIS4 and URA3 were at different locations within the insert, but displayed similar ratios between the two loci (data not shown). ( B ) Southern blots of Hin dIII digests of DNA from vde∆ strains, to detect total Spo11-initiated crossovers. ( C ) Southern blots of Hin dIII-VDE double digests of the same samples, to determine the background contribution of Spo11-initiated COs in subsequent experiments measuring VDE-initiated COs, which will be VDE-resistant due to conversion of the VRS site to VRS103 . Probes were as shown in Figure 1 . ( D ) Quantification of data in panels B (total COs; filled circles) and C (VDE-resistant COs; open circles). Data are from a single experiment. DOI: http://dx.doi.org/10.7554/eLife.19669.004

    Journal: eLife

    Article Title: Local chromosome context is a major determinant of crossover pathway biochemistry during budding yeast meiosis

    doi: 10.7554/eLife.19669

    Figure Lengend Snippet: Spo11-initiated events at the two insert loci. ( A ) Spo11-catalyzed DSBs are more frequent at HIS4 that at URA3 . Left—Southern blots of Eco RI digests of DNA from vde∆ strains, probed with pBR322 sequences, showing Spo11-DSBs in the Parent 2 insert (see Figure 1 ) in resection/repair-deficient sae2∆ mutant strains. Right—location of DSBs and probe and DSB frequencies (average of 7 and 8 hr samples from a single experiment; error bars represent range). Spo11-DSBs in the Parent 1 inserts at HIS4 and URA3 were at different locations within the insert, but displayed similar ratios between the two loci (data not shown). ( B ) Southern blots of Hin dIII digests of DNA from vde∆ strains, to detect total Spo11-initiated crossovers. ( C ) Southern blots of Hin dIII-VDE double digests of the same samples, to determine the background contribution of Spo11-initiated COs in subsequent experiments measuring VDE-initiated COs, which will be VDE-resistant due to conversion of the VRS site to VRS103 . Probes were as shown in Figure 1 . ( D ) Quantification of data in panels B (total COs; filled circles) and C (VDE-resistant COs; open circles). Data are from a single experiment. DOI: http://dx.doi.org/10.7554/eLife.19669.004

    Article Snippet: Recombination products were detected on Southern blots containing genomic DNA digested with Hin dIII and VDE (P I-Sce I, New England Biolabs), using specific buffer for P I-Sce I.

    Techniques: Mutagenesis

    70–80% of VDE-DSBs are repaired. ( A ) Fraction of inserts remaining, calculated using Hin dIII digests (see Figure 1 ). For the arg4-VRS103 insert, the ratio (Parent 2 + CO2)/ (0.5 x LC) was calculated at 9 hr, and was then normalized to the 0 hr value. For the arg4-VRS insert, a similar calculation was made: (Parent 1 + NCO + CO1)/(0.5 x LC) ( B ) Relative recovery of interhomolog recombination products, calculated using Hin dIII-VDE double digests (see Figure 1 ). The sum of CO (average of CO1 and CO2) and NCO frequencies was divided by the frequency of total DSBs, as calculated in Figure 2A . Data are the average of two independent experiments; error bars represent range. DOI: http://dx.doi.org/10.7554/eLife.19669.006

    Journal: eLife

    Article Title: Local chromosome context is a major determinant of crossover pathway biochemistry during budding yeast meiosis

    doi: 10.7554/eLife.19669

    Figure Lengend Snippet: 70–80% of VDE-DSBs are repaired. ( A ) Fraction of inserts remaining, calculated using Hin dIII digests (see Figure 1 ). For the arg4-VRS103 insert, the ratio (Parent 2 + CO2)/ (0.5 x LC) was calculated at 9 hr, and was then normalized to the 0 hr value. For the arg4-VRS insert, a similar calculation was made: (Parent 1 + NCO + CO1)/(0.5 x LC) ( B ) Relative recovery of interhomolog recombination products, calculated using Hin dIII-VDE double digests (see Figure 1 ). The sum of CO (average of CO1 and CO2) and NCO frequencies was divided by the frequency of total DSBs, as calculated in Figure 2A . Data are the average of two independent experiments; error bars represent range. DOI: http://dx.doi.org/10.7554/eLife.19669.006

    Article Snippet: Recombination products were detected on Southern blots containing genomic DNA digested with Hin dIII and VDE (P I-Sce I, New England Biolabs), using specific buffer for P I-Sce I.

    Techniques:

    Southern blots of Hin dIII and Hin dIII-VDE digests of DNA from HIS4 insert-containing strains (top) and from URA3 insert-contaning strains (bottom). Probes and gel labels are as in Figure 1 ; JM—joint molecule recombination intermediates. DOI: http://dx.doi.org/10.7554/eLife.19669.009

    Journal: eLife

    Article Title: Local chromosome context is a major determinant of crossover pathway biochemistry during budding yeast meiosis

    doi: 10.7554/eLife.19669

    Figure Lengend Snippet: Southern blots of Hin dIII and Hin dIII-VDE digests of DNA from HIS4 insert-containing strains (top) and from URA3 insert-contaning strains (bottom). Probes and gel labels are as in Figure 1 ; JM—joint molecule recombination intermediates. DOI: http://dx.doi.org/10.7554/eLife.19669.009

    Article Snippet: Recombination products were detected on Southern blots containing genomic DNA digested with Hin dIII and VDE (P I-Sce I, New England Biolabs), using specific buffer for P I-Sce I.

    Techniques:

    Southern blots of Hin dIII and Hin dIII-VDE digests of DNA from HIS4 insert-containing strains (top) and from URA3 insert-contaning strains (bottom). Gel labels are as in Figure 1 ; JM—joint molecule recombination intermediates. In the gel with Hin DIII digests of samples from a pch2∆ mm4-mn yen1∆ slx1∆ strain with inserts at URA3 , the 9 hr sample was originally loaded between the 4 and 5 hr samples; this image was cut and spliced as indicated by vertical lines for presentation purposes. DOI: http://dx.doi.org/10.7554/eLife.19669.012

    Journal: eLife

    Article Title: Local chromosome context is a major determinant of crossover pathway biochemistry during budding yeast meiosis

    doi: 10.7554/eLife.19669

    Figure Lengend Snippet: Southern blots of Hin dIII and Hin dIII-VDE digests of DNA from HIS4 insert-containing strains (top) and from URA3 insert-contaning strains (bottom). Gel labels are as in Figure 1 ; JM—joint molecule recombination intermediates. In the gel with Hin DIII digests of samples from a pch2∆ mm4-mn yen1∆ slx1∆ strain with inserts at URA3 , the 9 hr sample was originally loaded between the 4 and 5 hr samples; this image was cut and spliced as indicated by vertical lines for presentation purposes. DOI: http://dx.doi.org/10.7554/eLife.19669.012

    Article Snippet: Recombination products were detected on Southern blots containing genomic DNA digested with Hin dIII and VDE (P I-Sce I, New England Biolabs), using specific buffer for P I-Sce I.

    Techniques:

    hDNA2 D277A unwinds plasmid- and oligonucleotide-based DNA substrates. ( A ) Representative 1% agarose gel showing hDNA2 D277A helicase activity on a λDNA/HindIII substrate in a time-course experiment with 346 nM hRPA. Heat, heat-denatured DNA substrate. ( B ) Representative 1% agarose gel showing that nuclease- and helicase-deficient hDNA2 D277A K654R (lanes 2–6) and helicase-deficient hDNA2 K654R (lane 8) do not exhibit helicase activity. Lane 7, DNA unwinding by nuclease-deficient DNA2 D277A. Reactions contained 215 nM hRPA. ( C – E ) Representative 10% polyacrylamide gels showing the helicase activity of hDNA2 D277A with ( C ) 5’ overhang, ( D ) 3’ overhang and with ( E ) dsDNA substrates. Reactions contained 7.5 nM RPA. Heat, heat-denatured DNA substrate. ( F ) Representative 1% agarose gels showing DNA unwinding of a 2.7 kbp-long substrate by either hDNA2 D277A (left part, at 37°C) or yDna2 E675A (right part, at 30°C) in a kinetic experiment with 215 nM human RPA or 267 nM yeast RPA respectively. ( G ) Quantitation of experiments such as shown in F. Averages shown, n = 2; error bars, SEM. DOI: http://dx.doi.org/10.7554/eLife.18574.007

    Journal: eLife

    Article Title: Human DNA2 possesses a cryptic DNA unwinding activity that functionally integrates with BLM or WRN helicases

    doi: 10.7554/eLife.18574

    Figure Lengend Snippet: hDNA2 D277A unwinds plasmid- and oligonucleotide-based DNA substrates. ( A ) Representative 1% agarose gel showing hDNA2 D277A helicase activity on a λDNA/HindIII substrate in a time-course experiment with 346 nM hRPA. Heat, heat-denatured DNA substrate. ( B ) Representative 1% agarose gel showing that nuclease- and helicase-deficient hDNA2 D277A K654R (lanes 2–6) and helicase-deficient hDNA2 K654R (lane 8) do not exhibit helicase activity. Lane 7, DNA unwinding by nuclease-deficient DNA2 D277A. Reactions contained 215 nM hRPA. ( C – E ) Representative 10% polyacrylamide gels showing the helicase activity of hDNA2 D277A with ( C ) 5’ overhang, ( D ) 3’ overhang and with ( E ) dsDNA substrates. Reactions contained 7.5 nM RPA. Heat, heat-denatured DNA substrate. ( F ) Representative 1% agarose gels showing DNA unwinding of a 2.7 kbp-long substrate by either hDNA2 D277A (left part, at 37°C) or yDna2 E675A (right part, at 30°C) in a kinetic experiment with 215 nM human RPA or 267 nM yeast RPA respectively. ( G ) Quantitation of experiments such as shown in F. Averages shown, n = 2; error bars, SEM. DOI: http://dx.doi.org/10.7554/eLife.18574.007

    Article Snippet: The PCR products were digested with BamHI and HindIII restriction endonucleases (New England Biolabs, Ipswich, MA) and ligated into a pFastBac1 vector (Invitrogen, Carlsbad, CA) generating pFB-His-hDNA2-FLAG.

    Techniques: Plasmid Preparation, Agarose Gel Electrophoresis, Activity Assay, Recombinase Polymerase Amplification, Quantitation Assay

    Generation of floxed mice by the enhanced PITCh system. a Targeting strategy for the generation of flox Col12a1 mice by the enhanced PITCh system. Purple highlights indicate microhomologies between endogenous Col12a1 locus and PITCh-donor. Blue characters indicate CRISPR target sequences. Red characters indicate protospacer adjacent motif (PAM) sequences. Yellow lightnings indicate DSB sites. b Schematic diagram of pronuclear injection of Cas9 protein, Col12a1 -left, -right, and gRNA-s1 crRNAs, tracrRNA, PITCh-donor, and Exo1 mRNA. The red, purple, and blue boxes indicate the insert, Col12a1 microhomologies, and gRNA-s1 target sequences, respectively. c PCR screenings of newborns. d PCR-RFLP (restriction fragment length polymorphism) screenings of floxed newborn mice. e Summary of flox Col12a1 mouse production by the enhanced PITCh system. f Sequences of boundaries between Col12a1 and LoxPs. Blue, green, and red characters indicate microhomologies, HindIII sites, and LoxPs, respectively. g in vitro Cre-recombination assay. Cloned PCR products of flox alleles from three flox Col12a1 mice and genomic PCR of wildtype were incubated with or without Cre-recombinase. LF: left forward primer, RR: right reverse primer, MH: microhomology, M: molecular marker, and WT: wildtype

    Journal: BMC Genomics

    Article Title: Gene cassette knock-in in mammalian cells and zygotes by enhanced MMEJ

    doi: 10.1186/s12864-016-3331-9

    Figure Lengend Snippet: Generation of floxed mice by the enhanced PITCh system. a Targeting strategy for the generation of flox Col12a1 mice by the enhanced PITCh system. Purple highlights indicate microhomologies between endogenous Col12a1 locus and PITCh-donor. Blue characters indicate CRISPR target sequences. Red characters indicate protospacer adjacent motif (PAM) sequences. Yellow lightnings indicate DSB sites. b Schematic diagram of pronuclear injection of Cas9 protein, Col12a1 -left, -right, and gRNA-s1 crRNAs, tracrRNA, PITCh-donor, and Exo1 mRNA. The red, purple, and blue boxes indicate the insert, Col12a1 microhomologies, and gRNA-s1 target sequences, respectively. c PCR screenings of newborns. d PCR-RFLP (restriction fragment length polymorphism) screenings of floxed newborn mice. e Summary of flox Col12a1 mouse production by the enhanced PITCh system. f Sequences of boundaries between Col12a1 and LoxPs. Blue, green, and red characters indicate microhomologies, HindIII sites, and LoxPs, respectively. g in vitro Cre-recombination assay. Cloned PCR products of flox alleles from three flox Col12a1 mice and genomic PCR of wildtype were incubated with or without Cre-recombinase. LF: left forward primer, RR: right reverse primer, MH: microhomology, M: molecular marker, and WT: wildtype

    Article Snippet: For floxCol12a1 screening, PCR products were digested with HindIII (NEB).

    Techniques: Mouse Assay, CRISPR, Injection, Polymerase Chain Reaction, In Vitro, Recombination Assay, Clone Assay, Incubation, Marker