mwoi  (New England Biolabs)


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    Name:
    MwoI
    Description:
    MwoI 2 500 units
    Catalog Number:
    R0573L
    Price:
    290
    Category:
    Restriction Enzymes
    Size:
    2 500 units
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    Structured Review

    New England Biolabs mwoi
    MwoI
    MwoI 2 500 units
    https://www.bioz.com/result/mwoi/product/New England Biolabs
    Average 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mwoi - by Bioz Stars, 2021-05
    96/100 stars

    Images

    1) Product Images from "Hydrocephalus in a rat model of Meckel Gruber syndrome with a TMEM67 mutation"

    Article Title: Hydrocephalus in a rat model of Meckel Gruber syndrome with a TMEM67 mutation

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-37620-5

    Characterization of the TMEM67 genotype in the Wpk rat. ( a ) Design of dCAPs genotyping approach. Primers for nested PCR are labeled Nested F and Nested R. MwoI indicates the sequence and location of the MwoI site created during PCR with the upstream and downstream dCAPs primers (labeled Forward and Reverse, respectively). The WT genomic sequence is labeled WT and the mutant sequence is labeled TMEM67 −/− . The C → T mutation in the Wpk mutant is marked in red. ( b ) Polyacrylamide gel analysis of nested PCR products obtained from genomic DNA from (left to right) WT, heterozygous, or homozygous mutant rats. The expected product size was 157 bp. The left lane contains DNA length markers. Note that in the first lane to the left, a 50 bp DNA ladder (50–1350 bp) was used as a standard with 50 to 150 bp markers indicated ( c ) A polyacrylamide gel as in ( b ) except that the nested PCR products were amplified with the dCAPs primers (see a ). The expected product size was 51 bp. ( d ) A polyacrylamide gel as in ( c ) except that the dCAPs PCR products were digested with MwoI prior to electrophoresis. MwoI digestion of the dCAPS product from the WT allele is expected to produce two unresolved bands migrating slightly faster than the 30 bp marker. MwoI is not expected to cleave the dCAPs product from the mutant allele. Note that in the first lane from the left of both gels ( c , d ), a 10 bp DNA ladder (10–150) was used as a standard with the 20 to 50 bp markers indicated.
    Figure Legend Snippet: Characterization of the TMEM67 genotype in the Wpk rat. ( a ) Design of dCAPs genotyping approach. Primers for nested PCR are labeled Nested F and Nested R. MwoI indicates the sequence and location of the MwoI site created during PCR with the upstream and downstream dCAPs primers (labeled Forward and Reverse, respectively). The WT genomic sequence is labeled WT and the mutant sequence is labeled TMEM67 −/− . The C → T mutation in the Wpk mutant is marked in red. ( b ) Polyacrylamide gel analysis of nested PCR products obtained from genomic DNA from (left to right) WT, heterozygous, or homozygous mutant rats. The expected product size was 157 bp. The left lane contains DNA length markers. Note that in the first lane to the left, a 50 bp DNA ladder (50–1350 bp) was used as a standard with 50 to 150 bp markers indicated ( c ) A polyacrylamide gel as in ( b ) except that the nested PCR products were amplified with the dCAPs primers (see a ). The expected product size was 51 bp. ( d ) A polyacrylamide gel as in ( c ) except that the dCAPs PCR products were digested with MwoI prior to electrophoresis. MwoI digestion of the dCAPS product from the WT allele is expected to produce two unresolved bands migrating slightly faster than the 30 bp marker. MwoI is not expected to cleave the dCAPs product from the mutant allele. Note that in the first lane from the left of both gels ( c , d ), a 10 bp DNA ladder (10–150) was used as a standard with the 20 to 50 bp markers indicated.

    Techniques Used: Nested PCR, Labeling, Sequencing, Polymerase Chain Reaction, Mutagenesis, Amplification, Electrophoresis, Marker

    2) Product Images from "DICER1 Expression and Outcomes in Endometrioid Endometrial Adenocarcinoma"

    Article Title: DICER1 Expression and Outcomes in Endometrioid Endometrial Adenocarcinoma

    Journal: Cancer

    doi: 10.1002/cncr.25665

    Loss of heterozygosity analyses Panel A: PCR amplification products of marker rs1057035 in representative normal (N) / tumor (T) pairs digested with MwoI. Tumor 1870 demonstrates LOH (DICER1 expression = 2.38 arbitrary units). Panel B: PCR amplification products of marker rs1209904 in representative normal (N) / tumor (T) pairs digested with HpyCH4IV. Tumors 1641 (DICER1 expression = 12.29 arbitrary units), 1870 (DICER1 expression = 2.38 arbitrary units) and 1907 (DICER1 expression = 0.48 arbitrary units) demonstrate allelic imbalance. White arrows indicate the allelic fragment(s) with reduced intensity.
    Figure Legend Snippet: Loss of heterozygosity analyses Panel A: PCR amplification products of marker rs1057035 in representative normal (N) / tumor (T) pairs digested with MwoI. Tumor 1870 demonstrates LOH (DICER1 expression = 2.38 arbitrary units). Panel B: PCR amplification products of marker rs1209904 in representative normal (N) / tumor (T) pairs digested with HpyCH4IV. Tumors 1641 (DICER1 expression = 12.29 arbitrary units), 1870 (DICER1 expression = 2.38 arbitrary units) and 1907 (DICER1 expression = 0.48 arbitrary units) demonstrate allelic imbalance. White arrows indicate the allelic fragment(s) with reduced intensity.

    Techniques Used: Polymerase Chain Reaction, Amplification, Marker, Expressing

    3) Product Images from "Hydrocephalus in a rat model of Meckel Gruber syndrome with a TMEM67 mutation"

    Article Title: Hydrocephalus in a rat model of Meckel Gruber syndrome with a TMEM67 mutation

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-37620-5

    Characterization of the TMEM67 genotype in the Wpk rat. ( a ) Design of dCAPs genotyping approach. Primers for nested PCR are labeled Nested F and Nested R. MwoI indicates the sequence and location of the MwoI site created during PCR with the upstream and downstream dCAPs primers (labeled Forward and Reverse, respectively). The WT genomic sequence is labeled WT and the mutant sequence is labeled TMEM67 −/− . The C → T mutation in the Wpk mutant is marked in red. ( b ) Polyacrylamide gel analysis of nested PCR products obtained from genomic DNA from (left to right) WT, heterozygous, or homozygous mutant rats. The expected product size was 157 bp. The left lane contains DNA length markers. Note that in the first lane to the left, a 50 bp DNA ladder (50–1350 bp) was used as a standard with 50 to 150 bp markers indicated ( c ) A polyacrylamide gel as in ( b ) except that the nested PCR products were amplified with the dCAPs primers (see a ). The expected product size was 51 bp. ( d ) A polyacrylamide gel as in ( c ) except that the dCAPs PCR products were digested with MwoI prior to electrophoresis. MwoI digestion of the dCAPS product from the WT allele is expected to produce two unresolved bands migrating slightly faster than the 30 bp marker. MwoI is not expected to cleave the dCAPs product from the mutant allele. Note that in the first lane from the left of both gels ( c , d ), a 10 bp DNA ladder (10–150) was used as a standard with the 20 to 50 bp markers indicated.
    Figure Legend Snippet: Characterization of the TMEM67 genotype in the Wpk rat. ( a ) Design of dCAPs genotyping approach. Primers for nested PCR are labeled Nested F and Nested R. MwoI indicates the sequence and location of the MwoI site created during PCR with the upstream and downstream dCAPs primers (labeled Forward and Reverse, respectively). The WT genomic sequence is labeled WT and the mutant sequence is labeled TMEM67 −/− . The C → T mutation in the Wpk mutant is marked in red. ( b ) Polyacrylamide gel analysis of nested PCR products obtained from genomic DNA from (left to right) WT, heterozygous, or homozygous mutant rats. The expected product size was 157 bp. The left lane contains DNA length markers. Note that in the first lane to the left, a 50 bp DNA ladder (50–1350 bp) was used as a standard with 50 to 150 bp markers indicated ( c ) A polyacrylamide gel as in ( b ) except that the nested PCR products were amplified with the dCAPs primers (see a ). The expected product size was 51 bp. ( d ) A polyacrylamide gel as in ( c ) except that the dCAPs PCR products were digested with MwoI prior to electrophoresis. MwoI digestion of the dCAPS product from the WT allele is expected to produce two unresolved bands migrating slightly faster than the 30 bp marker. MwoI is not expected to cleave the dCAPs product from the mutant allele. Note that in the first lane from the left of both gels ( c , d ), a 10 bp DNA ladder (10–150) was used as a standard with the 20 to 50 bp markers indicated.

    Techniques Used: Nested PCR, Labeling, Sequencing, Polymerase Chain Reaction, Mutagenesis, Amplification, Electrophoresis, Marker

    Related Articles

    Amplification:

    Article Title: DICER1 Expression and Outcomes in Endometrioid Endometrial Adenocarcinoma
    Article Snippet: Sequences including rs1057035 and rs1209904 were PCR amplified in tumor and matched normal DNAs (rs1057035 Forward: 5′-AGTTAGGACTGCGGAAAGCA-3′ and Reverse: 5′-GCCGTTTCACTTTTTGTTGG-3′; rs1209904 Forward: 5′-TGCTGATCACACAGATCTTCAA-3′ and Reverse: 5′-GCTTTTGCCAAAGAAATTGG-3′). .. Amplification products were digested with MwoI and HpyCH4IV respectively (New England Biolabs, Ipswich, MA) and the resultant restriction digestion products resolved on 10% polyacrylamide gels. .. Methylation in the DICER1 5′ putative regulatory region was analyzed by combined bisulfate restriction analysis (COBRA) , .

    Polymerase Chain Reaction:

    Article Title: Rapid evolution of in vivo-selected sequences and structures replacing twenty percent of a subviral RNA
    Article Snippet: The 3′ fragment containing either 19, 38, or 76 random nt was generated by using respective forward primer 28-135randomize (5′-TAC GCAACTAATGC AGAACA[N19 or N38 or N76 ]AGTTCCCATCAA-3′) and reverse primer oligo 7 (5′-GGGCAGGCCCCCCGTCCGA-3′; complementary to 19 nt at the 3′ end of satC). .. PCR products were subjected to electrophoresis, purified using Wizard® SV Gel and PCR clean-up system columns (Promega, Madison, WI), digested with MwoI (all enzymes procured from New England Biolabs, Ipswich, MA, except where noted), phenol/chloroform extracted, and respective 5′ and 3′ fragments ligated together to produce full-length satC cDNA. .. These satC cDNAs with randomized wt or reduced length H2 were directly in vitro transcribed using T7 RNA polymerase.

    Article Title: Hydrocephalus in a rat model of Meckel Gruber syndrome with a TMEM67 mutation
    Article Snippet: The dCAPS PCR was performed in 20 μl reactions containing 2 μl of amplified products from the nested PCR with the following cycling conditions: 95 °C for 5 min (denaturation), followed by 30 cycles at 95 °C for 15 sec (denaturation), 44 °C for 15 sec (annealing), and 72 °C for 5 sec (extension). .. PCR products were digested with MwoI (New England Biolabs) in total reaction volumes of 50 μl by adding 10 μl of PCR product to 5 μl of the 10X CutSmart buffer (1:10 dilution) containing 5 units of MwoI (34 μl H2O, 10 μl PCR product, 5 μl 1X CutSmart buffer, and 1 μl MwoI). .. The samples were then incubated at 60 °C for 1 h. Following digestion, the samples were separated by electrophoresis on vertical 15% polyacrylamide gels in 1X Tris/boric acid/EDTA (TBE) buffer and visualized by staining with ethidium bromide.

    Article Title: PCR-Restriction Fragment Length Polymorphism Analysis of a Diagnostic 452-Base-Pair DNA Fragment Discriminates between Cryptosporidium parvum and C. meleagridis and between C. parvum Isolates of Human and Animal Origin
    Article Snippet: As our sequences, when aligned against the homologous C. wrairi sequence, showed polymorphism, a restriction fragment length polymorphism (RFLP)-based assay was developed. .. Purified PCR product was digested in a 20-μl mixture consisting of 1 U of Mwo I (New England Biolabs, Beverly, Mass.), 1 U of Mlu I (New England Biolabs), 1 U of Bpm I (New England Biolabs), 0.2 μl of 100× bovine serum albumin, and 2 μl of the appropriate 10× restriction buffer (NE buffer 3) under the conditions recommended by the supplier. .. The digestion mixture was incubated at 37°C for 2 h followed by 60°C for 2 h. The digest products were fractioned by 2% agarose gel electrophoresis and were visualized by ethidium bromide staining.

    Electrophoresis:

    Article Title: Rapid evolution of in vivo-selected sequences and structures replacing twenty percent of a subviral RNA
    Article Snippet: The 3′ fragment containing either 19, 38, or 76 random nt was generated by using respective forward primer 28-135randomize (5′-TAC GCAACTAATGC AGAACA[N19 or N38 or N76 ]AGTTCCCATCAA-3′) and reverse primer oligo 7 (5′-GGGCAGGCCCCCCGTCCGA-3′; complementary to 19 nt at the 3′ end of satC). .. PCR products were subjected to electrophoresis, purified using Wizard® SV Gel and PCR clean-up system columns (Promega, Madison, WI), digested with MwoI (all enzymes procured from New England Biolabs, Ipswich, MA, except where noted), phenol/chloroform extracted, and respective 5′ and 3′ fragments ligated together to produce full-length satC cDNA. .. These satC cDNAs with randomized wt or reduced length H2 were directly in vitro transcribed using T7 RNA polymerase.

    Purification:

    Article Title: Rapid evolution of in vivo-selected sequences and structures replacing twenty percent of a subviral RNA
    Article Snippet: The 3′ fragment containing either 19, 38, or 76 random nt was generated by using respective forward primer 28-135randomize (5′-TAC GCAACTAATGC AGAACA[N19 or N38 or N76 ]AGTTCCCATCAA-3′) and reverse primer oligo 7 (5′-GGGCAGGCCCCCCGTCCGA-3′; complementary to 19 nt at the 3′ end of satC). .. PCR products were subjected to electrophoresis, purified using Wizard® SV Gel and PCR clean-up system columns (Promega, Madison, WI), digested with MwoI (all enzymes procured from New England Biolabs, Ipswich, MA, except where noted), phenol/chloroform extracted, and respective 5′ and 3′ fragments ligated together to produce full-length satC cDNA. .. These satC cDNAs with randomized wt or reduced length H2 were directly in vitro transcribed using T7 RNA polymerase.

    Article Title: PCR-Restriction Fragment Length Polymorphism Analysis of a Diagnostic 452-Base-Pair DNA Fragment Discriminates between Cryptosporidium parvum and C. meleagridis and between C. parvum Isolates of Human and Animal Origin
    Article Snippet: As our sequences, when aligned against the homologous C. wrairi sequence, showed polymorphism, a restriction fragment length polymorphism (RFLP)-based assay was developed. .. Purified PCR product was digested in a 20-μl mixture consisting of 1 U of Mwo I (New England Biolabs, Beverly, Mass.), 1 U of Mlu I (New England Biolabs), 1 U of Bpm I (New England Biolabs), 0.2 μl of 100× bovine serum albumin, and 2 μl of the appropriate 10× restriction buffer (NE buffer 3) under the conditions recommended by the supplier. .. The digestion mixture was incubated at 37°C for 2 h followed by 60°C for 2 h. The digest products were fractioned by 2% agarose gel electrophoresis and were visualized by ethidium bromide staining.

    RFLP Assay:

    Article Title: A Molecular Epidemiological Survey of Clinically Important Dermatophytes in Iran Based on Specific RFLP Profiles of Beta-tubulin Gene
    Article Snippet: The following conditions were set up for amplification: initial denaturation phase at 94°C for 6 min, 35 cycles of 94°C for 30 sec, 58°C for 30 sec and 72°C for 1 min, followed by an ultimate extension step at 72°C for 10 min. .. Species identification was performed in a two-step RFLP assay, preliminary by using the restriction enzyme Fat I, (New England Biolabs Ltd, NHitchin, UK) and subsequently by Mwo I and Hpy CH4V (New England Biolabs Ltd, NHitchin, UK), and Alw 21I (Fermentas, Vilnius, Lithuania) according to the manufacturer’s instructions. .. All digestion reactions were performed in a 15 μl mixture containing 1.5 μl of 10 × buffer, 0.5 μl of each enzyme (5 U/μl), 8μl of BT2 amplicon and enough ultrapure water up to the final volume.

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    New England Biolabs mwoi
    Characterization of the TMEM67 genotype in the Wpk rat. ( a ) Design of dCAPs genotyping approach. Primers for nested <t>PCR</t> are labeled Nested F and Nested R. <t>MwoI</t> indicates the sequence and location of the MwoI site created during PCR with the upstream and downstream dCAPs primers (labeled Forward and Reverse, respectively). The WT genomic sequence is labeled WT and the mutant sequence is labeled TMEM67 −/− . The C → T mutation in the Wpk mutant is marked in red. ( b ) Polyacrylamide gel analysis of nested PCR products obtained from genomic DNA from (left to right) WT, heterozygous, or homozygous mutant rats. The expected product size was 157 bp. The left lane contains DNA length markers. Note that in the first lane to the left, a 50 bp DNA ladder (50–1350 bp) was used as a standard with 50 to 150 bp markers indicated ( c ) A polyacrylamide gel as in ( b ) except that the nested PCR products were amplified with the dCAPs primers (see a ). The expected product size was 51 bp. ( d ) A polyacrylamide gel as in ( c ) except that the dCAPs PCR products were digested with MwoI prior to electrophoresis. MwoI digestion of the dCAPS product from the WT allele is expected to produce two unresolved bands migrating slightly faster than the 30 bp marker. MwoI is not expected to cleave the dCAPs product from the mutant allele. Note that in the first lane from the left of both gels ( c , d ), a 10 bp DNA ladder (10–150) was used as a standard with the 20 to 50 bp markers indicated.
    Mwoi, 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
    https://www.bioz.com/result/mwoi/product/New England Biolabs
    Average 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mwoi - by Bioz Stars, 2021-05
    96/100 stars
      Buy from Supplier

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    Characterization of the TMEM67 genotype in the Wpk rat. ( a ) Design of dCAPs genotyping approach. Primers for nested PCR are labeled Nested F and Nested R. MwoI indicates the sequence and location of the MwoI site created during PCR with the upstream and downstream dCAPs primers (labeled Forward and Reverse, respectively). The WT genomic sequence is labeled WT and the mutant sequence is labeled TMEM67 −/− . The C → T mutation in the Wpk mutant is marked in red. ( b ) Polyacrylamide gel analysis of nested PCR products obtained from genomic DNA from (left to right) WT, heterozygous, or homozygous mutant rats. The expected product size was 157 bp. The left lane contains DNA length markers. Note that in the first lane to the left, a 50 bp DNA ladder (50–1350 bp) was used as a standard with 50 to 150 bp markers indicated ( c ) A polyacrylamide gel as in ( b ) except that the nested PCR products were amplified with the dCAPs primers (see a ). The expected product size was 51 bp. ( d ) A polyacrylamide gel as in ( c ) except that the dCAPs PCR products were digested with MwoI prior to electrophoresis. MwoI digestion of the dCAPS product from the WT allele is expected to produce two unresolved bands migrating slightly faster than the 30 bp marker. MwoI is not expected to cleave the dCAPs product from the mutant allele. Note that in the first lane from the left of both gels ( c , d ), a 10 bp DNA ladder (10–150) was used as a standard with the 20 to 50 bp markers indicated.

    Journal: Scientific Reports

    Article Title: Hydrocephalus in a rat model of Meckel Gruber syndrome with a TMEM67 mutation

    doi: 10.1038/s41598-018-37620-5

    Figure Lengend Snippet: Characterization of the TMEM67 genotype in the Wpk rat. ( a ) Design of dCAPs genotyping approach. Primers for nested PCR are labeled Nested F and Nested R. MwoI indicates the sequence and location of the MwoI site created during PCR with the upstream and downstream dCAPs primers (labeled Forward and Reverse, respectively). The WT genomic sequence is labeled WT and the mutant sequence is labeled TMEM67 −/− . The C → T mutation in the Wpk mutant is marked in red. ( b ) Polyacrylamide gel analysis of nested PCR products obtained from genomic DNA from (left to right) WT, heterozygous, or homozygous mutant rats. The expected product size was 157 bp. The left lane contains DNA length markers. Note that in the first lane to the left, a 50 bp DNA ladder (50–1350 bp) was used as a standard with 50 to 150 bp markers indicated ( c ) A polyacrylamide gel as in ( b ) except that the nested PCR products were amplified with the dCAPs primers (see a ). The expected product size was 51 bp. ( d ) A polyacrylamide gel as in ( c ) except that the dCAPs PCR products were digested with MwoI prior to electrophoresis. MwoI digestion of the dCAPS product from the WT allele is expected to produce two unresolved bands migrating slightly faster than the 30 bp marker. MwoI is not expected to cleave the dCAPs product from the mutant allele. Note that in the first lane from the left of both gels ( c , d ), a 10 bp DNA ladder (10–150) was used as a standard with the 20 to 50 bp markers indicated.

    Article Snippet: PCR products were digested with MwoI (New England Biolabs) in total reaction volumes of 50 μl by adding 10 μl of PCR product to 5 μl of the 10X CutSmart buffer (1:10 dilution) containing 5 units of MwoI (34 μl H2O, 10 μl PCR product, 5 μl 1X CutSmart buffer, and 1 μl MwoI).

    Techniques: Nested PCR, Labeling, Sequencing, Polymerase Chain Reaction, Mutagenesis, Amplification, Electrophoresis, Marker

    Loss of heterozygosity analyses Panel A: PCR amplification products of marker rs1057035 in representative normal (N) / tumor (T) pairs digested with MwoI. Tumor 1870 demonstrates LOH (DICER1 expression = 2.38 arbitrary units). Panel B: PCR amplification products of marker rs1209904 in representative normal (N) / tumor (T) pairs digested with HpyCH4IV. Tumors 1641 (DICER1 expression = 12.29 arbitrary units), 1870 (DICER1 expression = 2.38 arbitrary units) and 1907 (DICER1 expression = 0.48 arbitrary units) demonstrate allelic imbalance. White arrows indicate the allelic fragment(s) with reduced intensity.

    Journal: Cancer

    Article Title: DICER1 Expression and Outcomes in Endometrioid Endometrial Adenocarcinoma

    doi: 10.1002/cncr.25665

    Figure Lengend Snippet: Loss of heterozygosity analyses Panel A: PCR amplification products of marker rs1057035 in representative normal (N) / tumor (T) pairs digested with MwoI. Tumor 1870 demonstrates LOH (DICER1 expression = 2.38 arbitrary units). Panel B: PCR amplification products of marker rs1209904 in representative normal (N) / tumor (T) pairs digested with HpyCH4IV. Tumors 1641 (DICER1 expression = 12.29 arbitrary units), 1870 (DICER1 expression = 2.38 arbitrary units) and 1907 (DICER1 expression = 0.48 arbitrary units) demonstrate allelic imbalance. White arrows indicate the allelic fragment(s) with reduced intensity.

    Article Snippet: Amplification products were digested with MwoI and HpyCH4IV respectively (New England Biolabs, Ipswich, MA) and the resultant restriction digestion products resolved on 10% polyacrylamide gels.

    Techniques: Polymerase Chain Reaction, Amplification, Marker, Expressing

    Family pedigree and identification of the homozygous intervals and the mutation. ( A ) Pedigree: three patients (II-1, II-2 and II-7) were genotyped. The segregation of the PLEKHM 2 mutation that creates a Mwo I site is presented by the restriction analysis using this enzyme, under each individual. Digestion of the 687 bp amplicon resulted in 25, 73, 75, 83, 190 and 241 bp fragments; if the mutation is present, the 239 bp fragment is cleaved into 135 and 104 bp fragments. All patients were homozygous for the mutation; all available parents and all healthy siblings were heterozygotes, except sibling II-3 who was homozygous normal. ( B ) AgileMultiIdeogram showing the homozygous regions of chromosomes 1 and 11 shared among patients II-1, II-2 and II-7. The total length of the chromosomal homozygous regions was 15.3 Mb. ( C ) Sequence chromatogram of the PCR product of genomic DNA showing the single variant compatible as a causing mutation, a deletion of two nucleotides 1:16055171_16055172delAG (GRCh37/Hg 19) in exon 12. The chromatograms present the homozygous normal sequence (individual II-3), heterozygotes (Het) and mutant (Mut).

    Journal: Human Molecular Genetics

    Article Title: PLEKHM2 mutation leads to abnormal localization of lysosomes, impaired autophagy flux and associates with recessive dilated cardiomyopathy and left ventricular noncompaction

    doi: 10.1093/hmg/ddv423

    Figure Lengend Snippet: Family pedigree and identification of the homozygous intervals and the mutation. ( A ) Pedigree: three patients (II-1, II-2 and II-7) were genotyped. The segregation of the PLEKHM 2 mutation that creates a Mwo I site is presented by the restriction analysis using this enzyme, under each individual. Digestion of the 687 bp amplicon resulted in 25, 73, 75, 83, 190 and 241 bp fragments; if the mutation is present, the 239 bp fragment is cleaved into 135 and 104 bp fragments. All patients were homozygous for the mutation; all available parents and all healthy siblings were heterozygotes, except sibling II-3 who was homozygous normal. ( B ) AgileMultiIdeogram showing the homozygous regions of chromosomes 1 and 11 shared among patients II-1, II-2 and II-7. The total length of the chromosomal homozygous regions was 15.3 Mb. ( C ) Sequence chromatogram of the PCR product of genomic DNA showing the single variant compatible as a causing mutation, a deletion of two nucleotides 1:16055171_16055172delAG (GRCh37/Hg 19) in exon 12. The chromatograms present the homozygous normal sequence (individual II-3), heterozygotes (Het) and mutant (Mut).

    Article Snippet: Mwo I (NEB) digestion was done according to the manufacturer's instruction.

    Techniques: Mutagenesis, Amplification, Sequencing, Polymerase Chain Reaction, Variant Assay

    Characterization of the TMEM67 genotype in the Wpk rat. ( a ) Design of dCAPs genotyping approach. Primers for nested PCR are labeled Nested F and Nested R. MwoI indicates the sequence and location of the MwoI site created during PCR with the upstream and downstream dCAPs primers (labeled Forward and Reverse, respectively). The WT genomic sequence is labeled WT and the mutant sequence is labeled TMEM67 −/− . The C → T mutation in the Wpk mutant is marked in red. ( b ) Polyacrylamide gel analysis of nested PCR products obtained from genomic DNA from (left to right) WT, heterozygous, or homozygous mutant rats. The expected product size was 157 bp. The left lane contains DNA length markers. Note that in the first lane to the left, a 50 bp DNA ladder (50–1350 bp) was used as a standard with 50 to 150 bp markers indicated ( c ) A polyacrylamide gel as in ( b ) except that the nested PCR products were amplified with the dCAPs primers (see a ). The expected product size was 51 bp. ( d ) A polyacrylamide gel as in ( c ) except that the dCAPs PCR products were digested with MwoI prior to electrophoresis. MwoI digestion of the dCAPS product from the WT allele is expected to produce two unresolved bands migrating slightly faster than the 30 bp marker. MwoI is not expected to cleave the dCAPs product from the mutant allele. Note that in the first lane from the left of both gels ( c , d ), a 10 bp DNA ladder (10–150) was used as a standard with the 20 to 50 bp markers indicated.

    Journal: Scientific Reports

    Article Title: Hydrocephalus in a rat model of Meckel Gruber syndrome with a TMEM67 mutation

    doi: 10.1038/s41598-018-37620-5

    Figure Lengend Snippet: Characterization of the TMEM67 genotype in the Wpk rat. ( a ) Design of dCAPs genotyping approach. Primers for nested PCR are labeled Nested F and Nested R. MwoI indicates the sequence and location of the MwoI site created during PCR with the upstream and downstream dCAPs primers (labeled Forward and Reverse, respectively). The WT genomic sequence is labeled WT and the mutant sequence is labeled TMEM67 −/− . The C → T mutation in the Wpk mutant is marked in red. ( b ) Polyacrylamide gel analysis of nested PCR products obtained from genomic DNA from (left to right) WT, heterozygous, or homozygous mutant rats. The expected product size was 157 bp. The left lane contains DNA length markers. Note that in the first lane to the left, a 50 bp DNA ladder (50–1350 bp) was used as a standard with 50 to 150 bp markers indicated ( c ) A polyacrylamide gel as in ( b ) except that the nested PCR products were amplified with the dCAPs primers (see a ). The expected product size was 51 bp. ( d ) A polyacrylamide gel as in ( c ) except that the dCAPs PCR products were digested with MwoI prior to electrophoresis. MwoI digestion of the dCAPS product from the WT allele is expected to produce two unresolved bands migrating slightly faster than the 30 bp marker. MwoI is not expected to cleave the dCAPs product from the mutant allele. Note that in the first lane from the left of both gels ( c , d ), a 10 bp DNA ladder (10–150) was used as a standard with the 20 to 50 bp markers indicated.

    Article Snippet: PCR products were digested with MwoI (New England Biolabs) in total reaction volumes of 50 μl by adding 10 μl of PCR product to 5 μl of the 10X CutSmart buffer (1:10 dilution) containing 5 units of MwoI (34 μl H2O, 10 μl PCR product, 5 μl 1X CutSmart buffer, and 1 μl MwoI).

    Techniques: Nested PCR, Labeling, Sequencing, Polymerase Chain Reaction, Mutagenesis, Amplification, Electrophoresis, Marker