Genomic DNAs were isolated from all F2 individuals derived from crosses between ′Pungsannamul′ ′CWS5095′, and between ′Uram′ and ′CWS5095′. They were used for dCAPS analysis. The PCR primers (Table 2 ) were designed to detect a single-base substitution at nucleotide position 1127 of Sg-5 in ′CWS5095′. The nucleotide substitution (G to A ) generates a MboI site (GATC ) in the amplified PCR product from the wild parents. The PCR conditions were the same as those mentioned above. The amplified products were digested with MboI (Takara Bio Inc, Shiga, Japan) and separated on a 1.2% agarose gel.
Mbo 1
Manufactured by Takara Bio
Sourced in Japan
Mbo I is a type II restriction endonuclease enzyme that recognizes and cleaves the DNA sequence 5'-CTCAG-3' and its reverse complement 5'-GAGCT-3'.
Automatically generated - may contain errors
Lab products found in correlation
Hpaii, by Takara Bio (1 mentions)
4 6 diamidino 2 phenylindole dapi, by Agilent Technologies (1 mentions)
Dideoxy atp ddatp, by Jena Biosciences (1 mentions)
Dideoxy ttp ddttp, by Jena Biosciences (1 mentions)
Sau3ai, by Takara Bio (1 mentions)
Biotin 16 dutp, by Roche (1 mentions)
Digoxigenin 11 dutp, by Roche (1 mentions)
Brij 35, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Trizma base, by Merck Group (1 mentions)
Sybr green 1, by Thermo Fisher Scientific (1 mentions)
Hpych4iv, by New England Biolabs (1 mentions)
Hap 2, by Takara Bio (1 mentions)
Acc 2, by Takara Bio (1 mentions)
Afa 1, by Takara Bio (1 mentions)
Haeiii, by Takara Bio (1 mentions)
Nlaiii, by New England Biolabs (1 mentions)
Hinfi, by Takara Bio (1 mentions)
Taq pcr mastermix, by Tiangen Biotech (1 mentions)
5 protocols using mbo 1
1
Analyzing Allelic Variation in Soybean Sg-5 Gene
2
Histochemical Staining with DAB and DAPI
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Paraformaldehyde (PFA) was purchased from Merck (Darmstadt, Germany), and 3,3'-diaminobenzidine 4HCl (DAB) was purchased from Dojin Chemical Co. (Kumamoto , Japan). Bovine serum albumin (BSA) (essentially fatty acid and globulin-free), Trizma base and Brij-35 were from Sigma Chemical Co. (St. Louis, MO, USA). Biotin-16-dUTP, digoxigenin-11-dUTP and terminal deoxynucleotidyl transferase (TdT) were from Roche Diagnostics (Mannheim, Germany). Dideoxy ATP (ddATP) and dideoxy TTP (ddTTP) were from Jena Bioscience (Jena, Germany). Hpa II, Msp I, Sau3A I and Mbo I were purchased from Takara Bio Inc. (Shiga, Japan). 4',6-Diamidino-2-phenylindole (DAPI) was from DAKO (Glostrup, Denmark). Permount was purchased from Fisher Scientific Inc. (NJ, USA). All other reagents used in this study were from Wako Pure Chemicals (Osaka, Japan) and were of analytical grade.
3
DNA Extraction and Molecular Marker Development
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Total cellular DNA was extracted from fresh green leaves according to the procedure described by Roger and Bendich (1988) . Cleaved Amplified Polymorphic Sequence (CAPS) markers were developed as follows: using primers of Single Nucleotide Polymorphism (SNP) marker sets (Möhring et al. 2004) and Expressed Sequence Tag (EST) marker sets (Schneider et al. 1999 (link)), PCR products were generated, then digested with one of thirteen restriction endonucleases: Hae III, Hha I, Taq I, Hap II, Mbo I, Afa I, Xsp I, Alu I, Acc II (Takara Bio, Ohtsu, Japan), TspE I (TOYOBO, Osaka, Japan), Mse I, HpyCh4 IV and Nla III (New England BioLabs, Ipswich, MA) (Supplemental Table 1 ). Restriction digests were electrophoresed in 2% agarose gels. Thirty-two simple sequence repeat (SSR) markers used in this study were reported previously (Laurent et al. 2007 (link), McGrath et al. 2007 ) (Supplemental Table 1 ). Cycling parameters were 94°C for one min, 40 cycles of 50 or 60°C for one min, followed by one cycle at 72°C for 10 min. Amplified products were electrophoresed in a High Efficiency Genome Scanning (HEGS) system (Kikuchi et al. 2001) using a discontinuous non-denatured acrylamide gel and Tris/Borate/EDTA (TBE) buffer. Gels were scanned after staining with Sybr green I (Molecular Probes, Eugene, OR) and photographed under a UV trans-illuminator (ATTO, Tokyo, Japan).
4
Marine DNA Extraction and Characterization
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TIANGEN Marine Animal Tissue DNA Extraction Kit and 2× Taq PCR MasterMix were purchased from TIANGEN BIOTECH Co., Ltd. The DNA marker was purchased from Solarbio Science & Technology Co., Ltd. The restriction enzymes Eco147 I, Hinf I, Mbo I, Xag I, and Hind II were purchased from Takara Biomedical Technology Co., Ltd. Primer synthesis and sequencing were performed by Sangon Biotech Co., Ltd. The PCR machine used was the traditional T1 PCR instrument (Whatman Biometra). The gel imaging system used was INFINITY 3000 (Vilber Lourmat Sté). Spectrophotometer used was NanoPhotometer Pearl (Implen LLC, German).
5
Mitochondrial Genome Cloning and Mapping
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MtDNAs from I-12CMS(2) and I-12CMS(3) were partially digested with MboI (Takara Bio, Ohtsu, Japan) and fractionated on continuous sucrose density gradients (Sambrook et al. 1989) . Fragments of 15 to 20 kbp were collected and ligated into the BamHI-digested lambda DASH vector (Stratagene, La Jolla, CA). The DNA ligation mixture was packaged in vitro using Gigapack Gold (Stratagene). A total of 384 recombinant phages were randomly chosen from each of the resulting mtDNA libraries and used for a physical mapping study. DNA fragments located every 5 kbp on the TK-81mm-O (normal cytoplasm) mtDNA map (Kubo et al. 1995) , and several known mitochondrial gene sequences were used as probes for plaque hybridization.
DNA fragments at the extremities of the clone contigs were used to 'walk' the mitochondrial genome. All overlapping clones selected were tested by hybridization with several mitochondrial gene probes as well as with DNA fragments on the TK-81mm-O mtDNA map (Kubo et al. 1995) , to ensure that each clone was a true representation of the mtDNA sequences.
DNA fragments at the extremities of the clone contigs were used to 'walk' the mitochondrial genome. All overlapping clones selected were tested by hybridization with several mitochondrial gene probes as well as with DNA fragments on the TK-81mm-O mtDNA map (Kubo et al. 1995) , to ensure that each clone was a true representation of the mtDNA sequences.
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