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TaKaRa la pcr buffer ii
Southern blot and <t>PCR</t> analysis of GmKASIIA and GmKASIIB genes in the normal soybean cultivar Bay and three high-palmitic-acid mutants. (A) Hin d III-digested <t>DNA</t> fragments (lane 1, Bay; lane 2, KK7; lane 3, J10; lane 4, M22) were hybridized with a GmKASIIA cRNA probe. (B) GmKASIIA and GmKASIIB DNA fragments were amplified with gene-specific primer sets (lane 1, Bay; lane 2, KK7; lane 3, J10; lane 4, M22).
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1) Product Images from "Molecular characterization of two high-palmitic-acid mutant loci induced by X-ray irradiation in soybean"

Article Title: Molecular characterization of two high-palmitic-acid mutant loci induced by X-ray irradiation in soybean

Journal: Breeding Science

doi: 10.1270/jsbbs.61.631

Southern blot and PCR analysis of GmKASIIA and GmKASIIB genes in the normal soybean cultivar Bay and three high-palmitic-acid mutants. (A) Hin d III-digested DNA fragments (lane 1, Bay; lane 2, KK7; lane 3, J10; lane 4, M22) were hybridized with a GmKASIIA cRNA probe. (B) GmKASIIA and GmKASIIB DNA fragments were amplified with gene-specific primer sets (lane 1, Bay; lane 2, KK7; lane 3, J10; lane 4, M22).
Figure Legend Snippet: Southern blot and PCR analysis of GmKASIIA and GmKASIIB genes in the normal soybean cultivar Bay and three high-palmitic-acid mutants. (A) Hin d III-digested DNA fragments (lane 1, Bay; lane 2, KK7; lane 3, J10; lane 4, M22) were hybridized with a GmKASIIA cRNA probe. (B) GmKASIIA and GmKASIIB DNA fragments were amplified with gene-specific primer sets (lane 1, Bay; lane 2, KK7; lane 3, J10; lane 4, M22).

Techniques Used: Southern Blot, Polymerase Chain Reaction, Amplification

Development of PCR-based markers for the J10 and M22 mutations. (A) Locations of two primer sets for detecting the mutation in J10. N.D. means not detected. (B) Location of a primer set for detecting the mutation in M22. (C) Detection of the J10 mutation: Bay (lane 1), heterozygous (lane 2) and J10 (lane 3) DNA templates. (D) Detection of the M22 mutation: Bay (lane 1), heterozygous (lane 2) and M22 (lane 3) DNA templates.
Figure Legend Snippet: Development of PCR-based markers for the J10 and M22 mutations. (A) Locations of two primer sets for detecting the mutation in J10. N.D. means not detected. (B) Location of a primer set for detecting the mutation in M22. (C) Detection of the J10 mutation: Bay (lane 1), heterozygous (lane 2) and J10 (lane 3) DNA templates. (D) Detection of the M22 mutation: Bay (lane 1), heterozygous (lane 2) and M22 (lane 3) DNA templates.

Techniques Used: Polymerase Chain Reaction, Mutagenesis

2) Product Images from "Thermostable Mismatch-Recognizing Protein MutS Suppresses Nonspecific Amplification during Polymerase Chain Reaction (PCR)"

Article Title: Thermostable Mismatch-Recognizing Protein MutS Suppresses Nonspecific Amplification during Polymerase Chain Reaction (PCR)

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms14036436

Effects of Tth MutS on standard polymerase chain reaction (PCR) amplification of an 80-bp template. ( A ) Schema tic representation of the primers and templates used in ( B , C ). Perfectly matched, GT-mismatched or unpaired T-containing primers were used; ( B ) Perfectly matched (left), GT-mismatched (middle) or unpaired T-containing (right) primers were used to amplify the perfectly matched template; ( C ) The relative amounts of the products from perfectly matched (blue), GT-mismatched (red) or unpaired T-containing (purple) primers were plotted against the Tth MutS concentration for reactions using three polymerases: LA Taq (left), KOD polymerase (middle) and A. aeolicus DnaE (right). The amounts of the products were normalized by those at 0 μM Tth MutS.
Figure Legend Snippet: Effects of Tth MutS on standard polymerase chain reaction (PCR) amplification of an 80-bp template. ( A ) Schema tic representation of the primers and templates used in ( B , C ). Perfectly matched, GT-mismatched or unpaired T-containing primers were used; ( B ) Perfectly matched (left), GT-mismatched (middle) or unpaired T-containing (right) primers were used to amplify the perfectly matched template; ( C ) The relative amounts of the products from perfectly matched (blue), GT-mismatched (red) or unpaired T-containing (purple) primers were plotted against the Tth MutS concentration for reactions using three polymerases: LA Taq (left), KOD polymerase (middle) and A. aeolicus DnaE (right). The amounts of the products were normalized by those at 0 μM Tth MutS.

Techniques Used: Polymerase Chain Reaction, Amplification, Concentration Assay

Related Articles

Clone Assay:

Article Title: Interplay of a non-conjugative integrative element and a conjugative plasmid in the spread of antibiotic resistance via suicidal plasmid transfer from an aquaculture Vibrio isolate
Article Snippet: The PCR mixture (total volume, 10 μl) contained 20 pmol each of the primers, 1 μl of 10x LA PCR buffer II (Takara Bio Inc., Kusatsu, Japan), 4 mM each dNTP, 25 mM of MgCl2 , 0.5 U of LA Taq DNA polymerase (Takara Bio Inc.), and template DNA (20–50 ng). .. When necessary, PCR products were cloned into pGEM-T easy vector (Promega Corp., Madison, WI, USA) using Competent High DH5α competent cells (TOYOBO CO., LTD., Osaka, Japan).

Article Title: Molecular characterization of two high-palmitic-acid mutant loci induced by X-ray irradiation in soybean
Article Snippet: PCR was performed in a total volume of 20 μl, containing 50 ng DNA, 2 μL of 10× LA PCR buffer II (Takara), 400 μM of each dNTP, 0.5 μM of each primer and 1 unit of LA Taq DNA polymerase (Takara). .. The long PCR and RT-PCR (see next section) fragments were cloned into the pGEM vector by using a TA cloning kit (Promega) according to the manufacturer’s instructions.

Amplification:

Article Title: Cytogenomic identification and long-read single molecule real-time (SMRT) sequencing of a Bardet–Biedl Syndrome 9 (BBS9) deletion
Article Snippet: Long-read SMRT sequencing : Long-range PCR amplification across the identified breakpoint regions was accomplished using primers targeted to unique DNA sequences flanking the approximated deletion coordinates, and these amplicons were subjected to SMRTbell library construction and long-read SMRT sequencing (Pacific Biosciences, Menlo Park, CA). .. Long-range PCR reactions were performed in 50 µl containing ~100 ng of DNA, 1× LA PCR buffer II (TaKaRa), 0.4 µM of barcoded forward and reverse primers (Supplemental Table ), 0.4 mM dNTPs, 1 µL DMSO, and 2.5 units of TaKaRa LA Taq HS.

Article Title: Thermostable Mismatch-Recognizing Protein MutS Suppresses Nonspecific Amplification during Polymerase Chain Reaction (PCR)
Article Snippet: PCR was performed with 0.06 units/μL LA Taq Hot-Start Version (Takara, Shiga, Japan), 0.03 units/μL KOD polymerase (Toyobo, Tokyo, Japan) or 40 nM A. aeolicus DnaE, in 1× Takara LA PCR buffer II (Takara, Shiga, Japan) containing 20 nM template DNA; 400 nM primers; 400 μM dATP, dTTP, dCTP and dGTP and various concentrations of Tth MutS or Aae MutS. .. The abnormally low annealing temperature was used to permit extension of mismatched primers, which may explain the low efficiency of the PCR amplification in these experiments.

Article Title: Dispersal route of the Asian house rat (Rattus tanezumi) on mainland China: insights from microsatellite and mitochondrial DNA
Article Snippet: .. DNA was amplified in a thermocycler (SensoQuest, GER) in 25-μL reaction mixes containing 1 μL of total DNA (about 30 μg), 2.5 μL of 10× LA PCR Buffer II (Mg2+ Plus) (Takara, JPN), 2.5 mM of each dNTP (Takara, JPN), 10 pmol of each primer, and 0.75 U of LA Taq polymerase (Takara, JPN). ..

Article Title: Detection of the KIAA1549-BRAF fusion gene in cells forming microvascular proliferations in pilocytic astrocytoma
Article Snippet: PCR amplification was in a GeneAmp PCR System 9700 (Applied Biosystems, Waltham, MA, USA). .. Long and accurate PCR (LA-PCR) assays were performed in a total reaction volume of 50 μl containing 10 x LA-PCR Buffer II (5 μl, Takara Bio Inc., Shiga, Japan), a dNTP mixture (8 μl), distilled water (35.5 μl), 1 μl of 10 μM of each primer, and 1 μl of a genomic DNA sample.

Article Title: In silico guided reconstruction and analysis of ICAM-1-binding var genes from Plasmodium falciparum
Article Snippet: Each reaction comprised 10 × LA PCR Buffer II, magnesium chloride (MgCl2 ) at 2.5 mM final concentration, dNTP at 1 mM final concentration, primers at 0.3 mM final concentration each, 2.5 units of TaKaRa LA Taq ®, 2 µl template and sterile water up to 50 µl final volume. .. The DBLα tag was amplified from parasite cDNA using the primers DBLαAF′ and DBLαBR .

Article Title: Mitochondrial DNA 4977 bp deletion is a common phenomenon in hair and increases with age
Article Snippet: Paragraph title: DNA amplification ... PCR was performed in a 25 μL reacti on mixture containing 2.5 μL 10× LA PCR Buffer II (Mg2+ Plus) (TaKaRa Biotechnology, Japan), 200 μM dNTPs, 0.5 μ;M primers , 1.25 U Taq DNA polymerase, and 30 ng DNA template.

Article Title: Analysis of Chromosomal Numbers, Mitochondrial Genome, and Full-Length Transcriptome of Onychostoma brevibarba
Article Snippet: The amplifications were performed in a 25 μL reaction volume containing 1× LA PCR buffer II (Mg2+ ), 1.25 mM of dNTPs, 0.5 mM of each primer, 1.25 Unit of LA Taq polymerase (Takara, Dalian, China), and approximately 100 ng of template genomic DNA. .. The sequences homology and variation among the fragments amplified from this new species were analyzed using BioEdit (Hall ), ClustalW (Larkin et al. ), and MEGA7 (Kumar et al. ).

Synthesized:

Article Title: Thermostable Mismatch-Recognizing Protein MutS Suppresses Nonspecific Amplification during Polymerase Chain Reaction (PCR)
Article Snippet: PCR Using an 80-bp DNA Template DNAs were synthesized by BEX Co (Tokyo, Japan). .. PCR was performed with 0.06 units/μL LA Taq Hot-Start Version (Takara, Shiga, Japan), 0.03 units/μL KOD polymerase (Toyobo, Tokyo, Japan) or 40 nM A. aeolicus DnaE, in 1× Takara LA PCR buffer II (Takara, Shiga, Japan) containing 20 nM template DNA; 400 nM primers; 400 μM dATP, dTTP, dCTP and dGTP and various concentrations of Tth MutS or Aae MutS.

TA Cloning:

Article Title: Molecular characterization of two high-palmitic-acid mutant loci induced by X-ray irradiation in soybean
Article Snippet: PCR was performed in a total volume of 20 μl, containing 50 ng DNA, 2 μL of 10× LA PCR buffer II (Takara), 400 μM of each dNTP, 0.5 μM of each primer and 1 unit of LA Taq DNA polymerase (Takara). .. The long PCR and RT-PCR (see next section) fragments were cloned into the pGEM vector by using a TA cloning kit (Promega) according to the manufacturer’s instructions.

Quantitative RT-PCR:

Article Title: In silico guided reconstruction and analysis of ICAM-1-binding var genes from Plasmodium falciparum
Article Snippet: Each reaction comprised 10 × LA PCR Buffer II, magnesium chloride (MgCl2 ) at 2.5 mM final concentration, dNTP at 1 mM final concentration, primers at 0.3 mM final concentration each, 2.5 units of TaKaRa LA Taq ®, 2 µl template and sterile water up to 50 µl final volume. .. Reaction conditions were an initial denaturing step of 95 °C for 3 min, followed by 30 cycles of 95 °C for 30 sec, 47 °C for 30 sec, 65 °C for 30 sec, and a final extension of 65 °C for 3 min. 5′ UPS PCR was carried out using primers unique to the three main UPS types A, B and C in combination with a reverse DBLα specific primer designed for RT-qPCR (see Table ).

Electrophoresis:

Article Title: Detection of the KIAA1549-BRAF fusion gene in cells forming microvascular proliferations in pilocytic astrocytoma
Article Snippet: The PCR products were separated by electrophoresis on 2% agarose gels; the target bands were cut from the gels. .. Long and accurate PCR (LA-PCR) assays were performed in a total reaction volume of 50 μl containing 10 x LA-PCR Buffer II (5 μl, Takara Bio Inc., Shiga, Japan), a dNTP mixture (8 μl), distilled water (35.5 μl), 1 μl of 10 μM of each primer, and 1 μl of a genomic DNA sample.

Modification:

Article Title: Generation of Cashmere Goats Carrying an EDAR Gene Mutant Using CRISPR-Cas9-Mediated Genome Editing
Article Snippet: At 48 h post-transfection, the gDNAs of the modified and wild-type cells were extracted using the cell genome extraction kit (TianGen Biotech, Beijing, China). .. The resulting amplicons were purified and mixed with 10× La PCR Buffer II (TaKaRa Bio, Shiga, Japan), and a heteroduplex was formed by gradient annealing under the following conditions: 95°C for 10 min, 95 to 85°C ramping at -2°C/s, 85 to 25°C at -0.3°C/s, and a 4°C hold.

Electroporation:

Article Title: Generation of Cashmere Goats Carrying an EDAR Gene Mutant Using CRISPR-Cas9-Mediated Genome Editing
Article Snippet: These GFbs were transfected by electroporation, either with the sgRNA1 and Cas9 plasmids, or the sgRNA2 and Cas9 plasmids. .. The resulting amplicons were purified and mixed with 10× La PCR Buffer II (TaKaRa Bio, Shiga, Japan), and a heteroduplex was formed by gradient annealing under the following conditions: 95°C for 10 min, 95 to 85°C ramping at -2°C/s, 85 to 25°C at -0.3°C/s, and a 4°C hold.

Transfection:

Article Title: Generation of Cashmere Goats Carrying an EDAR Gene Mutant Using CRISPR-Cas9-Mediated Genome Editing
Article Snippet: These GFbs were transfected by electroporation, either with the sgRNA1 and Cas9 plasmids, or the sgRNA2 and Cas9 plasmids. .. The resulting amplicons were purified and mixed with 10× La PCR Buffer II (TaKaRa Bio, Shiga, Japan), and a heteroduplex was formed by gradient annealing under the following conditions: 95°C for 10 min, 95 to 85°C ramping at -2°C/s, 85 to 25°C at -0.3°C/s, and a 4°C hold.

Polymerase Chain Reaction:

Article Title: Cytogenomic identification and long-read single molecule real-time (SMRT) sequencing of a Bardet–Biedl Syndrome 9 (BBS9) deletion
Article Snippet: .. Long-range PCR reactions were performed in 50 µl containing ~100 ng of DNA, 1× LA PCR buffer II (TaKaRa), 0.4 µM of barcoded forward and reverse primers (Supplemental Table ), 0.4 mM dNTPs, 1 µL DMSO, and 2.5 units of TaKaRa LA Taq HS. .. Amplification consisted of an initial denaturation step at 95 °C for 5 min followed by 10 amplification cycles (95 °C for 30 s, 61 °C for 30 s, and 72 °C for 10 min), another 20 amplification cycles (95 °C for 30 s, 56 °C for 30 s, and 72 °C for 10 min), and a final extension at 72 °C for 15 min. All PCR amplicons were purified, quantified, pooled, sequenced (P6-C4 PacBio protocol), and analyzed as previously described.

Article Title: Molecular heterogeneity in the novel fusion gene APIP-FGFR2: Diversity of genomic breakpoints in gastric cancer with high-level amplifications at 11p13 and 10q26
Article Snippet: .. For the long-distance PCR, each reaction mixture (50 µl) contained 1 ng BAC DNA, 10 pmol of each primer, 8 µl of dNTP mixture (2.5 mM each), 5 µl LA PCR Buffer II and 2.5 U of Takara LA Taq HS (Takara Bio, Inc., Otsu, Japan). .. Reaction conditions were as follows: denaturation at 94°C for 1 min, followed by 30 cycles of denaturation at 98°C for 20 sec, annealing and extension at 68°C for 10 min with 15-sec increments per cycle, and a final extension at 72°C for 10 min. L4 contained exons 2–5 of APIP (nucleotides 118, 330-128, 595 in RP11-412L22), and L1 contained exons 11–13 of FGFR2 (nucleotides 27, 769-38, 427 in RP11-62L18).

Article Title: Thermostable Mismatch-Recognizing Protein MutS Suppresses Nonspecific Amplification during Polymerase Chain Reaction (PCR)
Article Snippet: .. PCR was performed with 0.06 units/μL LA Taq Hot-Start Version (Takara, Shiga, Japan), 0.03 units/μL KOD polymerase (Toyobo, Tokyo, Japan) or 40 nM A. aeolicus DnaE, in 1× Takara LA PCR buffer II (Takara, Shiga, Japan) containing 20 nM template DNA; 400 nM primers; 400 μM dATP, dTTP, dCTP and dGTP and various concentrations of Tth MutS or Aae MutS. .. Twenty PCR cycles were run using the temperature controlling system PC707 (Astec, Tokyo, Japan): denaturation step, 80 °C for 1 min; annealing, 48 °C for 1 min; and extension, 70 °C for 2 min (slope of 1 min).

Article Title: Dispersal route of the Asian house rat (Rattus tanezumi) on mainland China: insights from microsatellite and mitochondrial DNA
Article Snippet: .. DNA was amplified in a thermocycler (SensoQuest, GER) in 25-μL reaction mixes containing 1 μL of total DNA (about 30 μg), 2.5 μL of 10× LA PCR Buffer II (Mg2+ Plus) (Takara, JPN), 2.5 mM of each dNTP (Takara, JPN), 10 pmol of each primer, and 0.75 U of LA Taq polymerase (Takara, JPN). ..

Article Title: Interplay of a non-conjugative integrative element and a conjugative plasmid in the spread of antibiotic resistance via suicidal plasmid transfer from an aquaculture Vibrio isolate
Article Snippet: .. The PCR mixture (total volume, 10 μl) contained 20 pmol each of the primers, 1 μl of 10x LA PCR buffer II (Takara Bio Inc., Kusatsu, Japan), 4 mM each dNTP, 25 mM of MgCl2 , 0.5 U of LA Taq DNA polymerase (Takara Bio Inc.), and template DNA (20–50 ng). .. Two-step PCR was performed with 30 cycles of denaturation at 96°C for 20 sec and extension at 69°C for 16 min.

Article Title: Detection of the KIAA1549-BRAF fusion gene in cells forming microvascular proliferations in pilocytic astrocytoma
Article Snippet: .. Long and accurate PCR (LA-PCR) assays were performed in a total reaction volume of 50 μl containing 10 x LA-PCR Buffer II (5 μl, Takara Bio Inc., Shiga, Japan), a dNTP mixture (8 μl), distilled water (35.5 μl), 1 μl of 10 μM of each primer, and 1 μl of a genomic DNA sample. .. PCR amplification was in a GeneAmp PCR System 9700 (Applied Biosystems).

Article Title: In silico guided reconstruction and analysis of ICAM-1-binding var genes from Plasmodium falciparum
Article Snippet: .. Each reaction comprised 10 × LA PCR Buffer II, magnesium chloride (MgCl2 ) at 2.5 mM final concentration, dNTP at 1 mM final concentration, primers at 0.3 mM final concentration each, 2.5 units of TaKaRa LA Taq ®, 2 µl template and sterile water up to 50 µl final volume. .. The DBLα tag was amplified from parasite cDNA using the primers DBLαAF′ and DBLαBR .

Article Title: Mitochondrial DNA 4977 bp deletion is a common phenomenon in hair and increases with age
Article Snippet: .. PCR was performed in a 25 μL reacti on mixture containing 2.5 μL 10× LA PCR Buffer II (Mg2+ Plus) (TaKaRa Biotechnology, Japan), 200 μM dNTPs, 0.5 μ;M primers , 1.25 U Taq DNA polymerase, and 30 ng DNA template. .. After the reaction, 5 μL of the PCR product was separated on 2% agarose gel and visualized with SYBR Safe (Invitrogen, USA) with UV.

Article Title: Generation of Cashmere Goats Carrying an EDAR Gene Mutant Using CRISPR-Cas9-Mediated Genome Editing
Article Snippet: .. The resulting amplicons were purified and mixed with 10× La PCR Buffer II (TaKaRa Bio, Shiga, Japan), and a heteroduplex was formed by gradient annealing under the following conditions: 95°C for 10 min, 95 to 85°C ramping at -2°C/s, 85 to 25°C at -0.3°C/s, and a 4°C hold. .. The heteroduplexes were processed using the Surveyor Mutation Detection Kit (Transgenomic, Omaha, NE, USA) and run on a 2% agarose gel to detect mutation efficiency.

Article Title: Insulin resistance in cavefish as an adaptation to a nutrient-limited environment
Article Snippet: .. PCR reactions were carried out in 12.5-µl volume containing 1× LA PCR Buffer II (Clontech), 2.5 mM MgCl2 , 0.4 mM dNTP mix, 0.4 µM of each forward and reverse primer and 0.05 units of TaKaRa LA Taq DNA Polymerase (Clontech). .. The PCR cycling conditions were as follows: initial denaturation at 94 °C for 2 min, followed by 35 cycles of 94 °C for 30 s, annealing temperature 52 °C for 30 s and 72 °C for 1 min. A final 5-min elongation step was performed at 72 °C.

Article Title: Complete Unique Genome Sequence, Expression Profile, and Salivary Gland Tissue Tropism of the Herpesvirus 7 Homolog in Pigtailed Macaques
Article Snippet: .. The PCR buffer consisted of the LA PCR buffer II (10×, Mg; TaKaRa) supplemented with 5% dimethyl sulfoxide. ..

Article Title: Molecular characterization of two high-palmitic-acid mutant loci induced by X-ray irradiation in soybean
Article Snippet: .. PCR was performed in a total volume of 20 μl, containing 50 ng DNA, 2 μL of 10× LA PCR buffer II (Takara), 400 μM of each dNTP, 0.5 μM of each primer and 1 unit of LA Taq DNA polymerase (Takara). ..

Article Title: Analysis of Chromosomal Numbers, Mitochondrial Genome, and Full-Length Transcriptome of Onychostoma brevibarba
Article Snippet: .. The amplifications were performed in a 25 μL reaction volume containing 1× LA PCR buffer II (Mg2+ ), 1.25 mM of dNTPs, 0.5 mM of each primer, 1.25 Unit of LA Taq polymerase (Takara, Dalian, China), and approximately 100 ng of template genomic DNA. ..

Sequencing:

Article Title: Cytogenomic identification and long-read single molecule real-time (SMRT) sequencing of a Bardet–Biedl Syndrome 9 (BBS9) deletion
Article Snippet: Long-read SMRT sequencing : Long-range PCR amplification across the identified breakpoint regions was accomplished using primers targeted to unique DNA sequences flanking the approximated deletion coordinates, and these amplicons were subjected to SMRTbell library construction and long-read SMRT sequencing (Pacific Biosciences, Menlo Park, CA). .. Long-range PCR reactions were performed in 50 µl containing ~100 ng of DNA, 1× LA PCR buffer II (TaKaRa), 0.4 µM of barcoded forward and reverse primers (Supplemental Table ), 0.4 mM dNTPs, 1 µL DMSO, and 2.5 units of TaKaRa LA Taq HS.

Article Title: Interplay of a non-conjugative integrative element and a conjugative plasmid in the spread of antibiotic resistance via suicidal plasmid transfer from an aquaculture Vibrio isolate
Article Snippet: The PCR mixture (total volume, 10 μl) contained 20 pmol each of the primers, 1 μl of 10x LA PCR buffer II (Takara Bio Inc., Kusatsu, Japan), 4 mM each dNTP, 25 mM of MgCl2 , 0.5 U of LA Taq DNA polymerase (Takara Bio Inc.), and template DNA (20–50 ng). .. Sanger sequencing was performed using BigDye v 3.1 (Thermo Fisher Scientific, Waltham, MA, USA) and ABI 3130xl Genetic Analyzer (Thermo Fisher Scientific).

Article Title: Generation of Cashmere Goats Carrying an EDAR Gene Mutant Using CRISPR-Cas9-Mediated Genome Editing
Article Snippet: These gDNAs were then used as templates to amplify the target sequence of the EDAR gene via PCR with the following primers 5′-GTGGTGGTCGTCGTGGTGATGC-3′ (forward) and 5′-CTGCTCAGCCTTCCTTATGGTC-3′ (reverse). .. The resulting amplicons were purified and mixed with 10× La PCR Buffer II (TaKaRa Bio, Shiga, Japan), and a heteroduplex was formed by gradient annealing under the following conditions: 95°C for 10 min, 95 to 85°C ramping at -2°C/s, 85 to 25°C at -0.3°C/s, and a 4°C hold.

Article Title: Insulin resistance in cavefish as an adaptation to a nutrient-limited environment
Article Snippet: PCR reactions were carried out in 12.5-µl volume containing 1× LA PCR Buffer II (Clontech), 2.5 mM MgCl2 , 0.4 mM dNTP mix, 0.4 µM of each forward and reverse primer and 0.05 units of TaKaRa LA Taq DNA Polymerase (Clontech). .. The PCR products were diluted tenfold and sequenced directly on a 3730XL DNA Analyzer (Applied Biosystems) using the sequencing primer: GGTGGAGTTGATGGTGGTATAG.

Article Title: Complete Unique Genome Sequence, Expression Profile, and Salivary Gland Tissue Tropism of the Herpesvirus 7 Homolog in Pigtailed Macaques
Article Snippet: Paragraph title: Long-range sequencing. ... The PCR buffer consisted of the LA PCR buffer II (10×, Mg; TaKaRa) supplemented with 5% dimethyl sulfoxide.

Article Title: Molecular characterization of two high-palmitic-acid mutant loci induced by X-ray irradiation in soybean
Article Snippet: Paragraph title: Long PCR and nucleotide sequencing ... PCR was performed in a total volume of 20 μl, containing 50 ng DNA, 2 μL of 10× LA PCR buffer II (Takara), 400 μM of each dNTP, 0.5 μM of each primer and 1 unit of LA Taq DNA polymerase (Takara).

Article Title: Analysis of Chromosomal Numbers, Mitochondrial Genome, and Full-Length Transcriptome of Onychostoma brevibarba
Article Snippet: Ten pairs of polymerase chain reaction (PCR) primers (Supplementary Table ) were designed to amplify the entire mitogenome sequence based on the conserved sequences of Cyprinid species retrieved from GenBank using Primer 5.0 software. .. The amplifications were performed in a 25 μL reaction volume containing 1× LA PCR buffer II (Mg2+ ), 1.25 mM of dNTPs, 0.5 mM of each primer, 1.25 Unit of LA Taq polymerase (Takara, Dalian, China), and approximately 100 ng of template genomic DNA.

Fluorescence:

Article Title: Molecular heterogeneity in the novel fusion gene APIP-FGFR2: Diversity of genomic breakpoints in gastric cancer with high-level amplifications at 11p13 and 10q26
Article Snippet: Paragraph title: Fluorescence in situ hybridization (FISH) analysis ... For the long-distance PCR, each reaction mixture (50 µl) contained 1 ng BAC DNA, 10 pmol of each primer, 8 µl of dNTP mixture (2.5 mM each), 5 µl LA PCR Buffer II and 2.5 U of Takara LA Taq HS (Takara Bio, Inc., Otsu, Japan).

Mutagenesis:

Article Title: Generation of Cashmere Goats Carrying an EDAR Gene Mutant Using CRISPR-Cas9-Mediated Genome Editing
Article Snippet: Paragraph title: Surveyor nuclease mutation-detection assay ... The resulting amplicons were purified and mixed with 10× La PCR Buffer II (TaKaRa Bio, Shiga, Japan), and a heteroduplex was formed by gradient annealing under the following conditions: 95°C for 10 min, 95 to 85°C ramping at -2°C/s, 85 to 25°C at -0.3°C/s, and a 4°C hold.

Size-exclusion Chromatography:

Article Title: Molecular heterogeneity in the novel fusion gene APIP-FGFR2: Diversity of genomic breakpoints in gastric cancer with high-level amplifications at 11p13 and 10q26
Article Snippet: For the long-distance PCR, each reaction mixture (50 µl) contained 1 ng BAC DNA, 10 pmol of each primer, 8 µl of dNTP mixture (2.5 mM each), 5 µl LA PCR Buffer II and 2.5 U of Takara LA Taq HS (Takara Bio, Inc., Otsu, Japan). .. Reaction conditions were as follows: denaturation at 94°C for 1 min, followed by 30 cycles of denaturation at 98°C for 20 sec, annealing and extension at 68°C for 10 min with 15-sec increments per cycle, and a final extension at 72°C for 10 min. L4 contained exons 2–5 of APIP (nucleotides 118, 330-128, 595 in RP11-412L22), and L1 contained exons 11–13 of FGFR2 (nucleotides 27, 769-38, 427 in RP11-62L18).

Article Title: Interplay of a non-conjugative integrative element and a conjugative plasmid in the spread of antibiotic resistance via suicidal plasmid transfer from an aquaculture Vibrio isolate
Article Snippet: The PCR mixture (total volume, 10 μl) contained 20 pmol each of the primers, 1 μl of 10x LA PCR buffer II (Takara Bio Inc., Kusatsu, Japan), 4 mM each dNTP, 25 mM of MgCl2 , 0.5 U of LA Taq DNA polymerase (Takara Bio Inc.), and template DNA (20–50 ng). .. Two-step PCR was performed with 30 cycles of denaturation at 96°C for 20 sec and extension at 69°C for 16 min.

Article Title: Detection of the KIAA1549-BRAF fusion gene in cells forming microvascular proliferations in pilocytic astrocytoma
Article Snippet: The thermal cycling conditions were: initial denaturation (95°C, 2 min), 35 cycles of denaturation (95°C, 30 sec), annealing (58°C, 30 sec), extension (72°C, 20 sec), final extension (72°C, 5 min). .. Long and accurate PCR (LA-PCR) assays were performed in a total reaction volume of 50 μl containing 10 x LA-PCR Buffer II (5 μl, Takara Bio Inc., Shiga, Japan), a dNTP mixture (8 μl), distilled water (35.5 μl), 1 μl of 10 μM of each primer, and 1 μl of a genomic DNA sample.

Article Title: In silico guided reconstruction and analysis of ICAM-1-binding var genes from Plasmodium falciparum
Article Snippet: Each reaction comprised 10 × LA PCR Buffer II, magnesium chloride (MgCl2 ) at 2.5 mM final concentration, dNTP at 1 mM final concentration, primers at 0.3 mM final concentration each, 2.5 units of TaKaRa LA Taq ®, 2 µl template and sterile water up to 50 µl final volume. .. Reaction conditions were an initial denaturing step of 95 °C for 3 min, followed by 30 cycles of 95 °C for 30 sec, 47 °C for 30 sec, 65 °C for 30 sec, and a final extension of 65 °C for 3 min. 5′ UPS PCR was carried out using primers unique to the three main UPS types A, B and C in combination with a reverse DBLα specific primer designed for RT-qPCR (see Table ).

Purification:

Article Title: Cytogenomic identification and long-read single molecule real-time (SMRT) sequencing of a Bardet–Biedl Syndrome 9 (BBS9) deletion
Article Snippet: Long-range PCR reactions were performed in 50 µl containing ~100 ng of DNA, 1× LA PCR buffer II (TaKaRa), 0.4 µM of barcoded forward and reverse primers (Supplemental Table ), 0.4 mM dNTPs, 1 µL DMSO, and 2.5 units of TaKaRa LA Taq HS. .. Amplification consisted of an initial denaturation step at 95 °C for 5 min followed by 10 amplification cycles (95 °C for 30 s, 61 °C for 30 s, and 72 °C for 10 min), another 20 amplification cycles (95 °C for 30 s, 56 °C for 30 s, and 72 °C for 10 min), and a final extension at 72 °C for 15 min. All PCR amplicons were purified, quantified, pooled, sequenced (P6-C4 PacBio protocol), and analyzed as previously described.

Article Title: Generation of Cashmere Goats Carrying an EDAR Gene Mutant Using CRISPR-Cas9-Mediated Genome Editing
Article Snippet: .. The resulting amplicons were purified and mixed with 10× La PCR Buffer II (TaKaRa Bio, Shiga, Japan), and a heteroduplex was formed by gradient annealing under the following conditions: 95°C for 10 min, 95 to 85°C ramping at -2°C/s, 85 to 25°C at -0.3°C/s, and a 4°C hold. .. The heteroduplexes were processed using the Surveyor Mutation Detection Kit (Transgenomic, Omaha, NE, USA) and run on a 2% agarose gel to detect mutation efficiency.

Article Title: Analysis of Chromosomal Numbers, Mitochondrial Genome, and Full-Length Transcriptome of Onychostoma brevibarba
Article Snippet: The amplifications were performed in a 25 μL reaction volume containing 1× LA PCR buffer II (Mg2+ ), 1.25 mM of dNTPs, 0.5 mM of each primer, 1.25 Unit of LA Taq polymerase (Takara, Dalian, China), and approximately 100 ng of template genomic DNA. .. Subsequently, the targeted fragments were purified using a gel extraction kit (Sangon, Shanghai, China) and directly sequenced by the Sangon Biotechnology Company (Sangon, Shanghai, China).

Reverse Transcription Polymerase Chain Reaction:

Article Title: Detection of the KIAA1549-BRAF fusion gene in cells forming microvascular proliferations in pilocytic astrocytoma
Article Snippet: Polymerase chain reaction (PCR) Reverse transcription PCR (RT-PCR) assays were performed in a total reaction volume of 25 μl containing 12.5 μl Go Taq HS (Promega, Madison, WI, USA), 9.5 μl distilled water, 1 μl of 10 μM of each primer, and 1 μl of cDNA- or genomic DNA samples. .. Long and accurate PCR (LA-PCR) assays were performed in a total reaction volume of 50 μl containing 10 x LA-PCR Buffer II (5 μl, Takara Bio Inc., Shiga, Japan), a dNTP mixture (8 μl), distilled water (35.5 μl), 1 μl of 10 μM of each primer, and 1 μl of a genomic DNA sample.

Article Title: Molecular characterization of two high-palmitic-acid mutant loci induced by X-ray irradiation in soybean
Article Snippet: PCR was performed in a total volume of 20 μl, containing 50 ng DNA, 2 μL of 10× LA PCR buffer II (Takara), 400 μM of each dNTP, 0.5 μM of each primer and 1 unit of LA Taq DNA polymerase (Takara). .. The long PCR and RT-PCR (see next section) fragments were cloned into the pGEM vector by using a TA cloning kit (Promega) according to the manufacturer’s instructions.

In Situ Hybridization:

Article Title: Molecular heterogeneity in the novel fusion gene APIP-FGFR2: Diversity of genomic breakpoints in gastric cancer with high-level amplifications at 11p13 and 10q26
Article Snippet: Paragraph title: Fluorescence in situ hybridization (FISH) analysis ... For the long-distance PCR, each reaction mixture (50 µl) contained 1 ng BAC DNA, 10 pmol of each primer, 8 µl of dNTP mixture (2.5 mM each), 5 µl LA PCR Buffer II and 2.5 U of Takara LA Taq HS (Takara Bio, Inc., Otsu, Japan).

Plasmid Preparation:

Article Title: Interplay of a non-conjugative integrative element and a conjugative plasmid in the spread of antibiotic resistance via suicidal plasmid transfer from an aquaculture Vibrio isolate
Article Snippet: The PCR mixture (total volume, 10 μl) contained 20 pmol each of the primers, 1 μl of 10x LA PCR buffer II (Takara Bio Inc., Kusatsu, Japan), 4 mM each dNTP, 25 mM of MgCl2 , 0.5 U of LA Taq DNA polymerase (Takara Bio Inc.), and template DNA (20–50 ng). .. When necessary, PCR products were cloned into pGEM-T easy vector (Promega Corp., Madison, WI, USA) using Competent High DH5α competent cells (TOYOBO CO., LTD., Osaka, Japan).

Article Title: Molecular characterization of two high-palmitic-acid mutant loci induced by X-ray irradiation in soybean
Article Snippet: PCR was performed in a total volume of 20 μl, containing 50 ng DNA, 2 μL of 10× LA PCR buffer II (Takara), 400 μM of each dNTP, 0.5 μM of each primer and 1 unit of LA Taq DNA polymerase (Takara). .. The long PCR and RT-PCR (see next section) fragments were cloned into the pGEM vector by using a TA cloning kit (Promega) according to the manufacturer’s instructions.

Software:

Article Title: Analysis of Chromosomal Numbers, Mitochondrial Genome, and Full-Length Transcriptome of Onychostoma brevibarba
Article Snippet: Ten pairs of polymerase chain reaction (PCR) primers (Supplementary Table ) were designed to amplify the entire mitogenome sequence based on the conserved sequences of Cyprinid species retrieved from GenBank using Primer 5.0 software. .. The amplifications were performed in a 25 μL reaction volume containing 1× LA PCR buffer II (Mg2+ ), 1.25 mM of dNTPs, 0.5 mM of each primer, 1.25 Unit of LA Taq polymerase (Takara, Dalian, China), and approximately 100 ng of template genomic DNA.

Agarose Gel Electrophoresis:

Article Title: Mitochondrial DNA 4977 bp deletion is a common phenomenon in hair and increases with age
Article Snippet: PCR was performed in a 25 μL reacti on mixture containing 2.5 μL 10× LA PCR Buffer II (Mg2+ Plus) (TaKaRa Biotechnology, Japan), 200 μM dNTPs, 0.5 μ;M primers , 1.25 U Taq DNA polymerase, and 30 ng DNA template. .. After the reaction, 5 μL of the PCR product was separated on 2% agarose gel and visualized with SYBR Safe (Invitrogen, USA) with UV.

Article Title: Generation of Cashmere Goats Carrying an EDAR Gene Mutant Using CRISPR-Cas9-Mediated Genome Editing
Article Snippet: The resulting amplicons were purified and mixed with 10× La PCR Buffer II (TaKaRa Bio, Shiga, Japan), and a heteroduplex was formed by gradient annealing under the following conditions: 95°C for 10 min, 95 to 85°C ramping at -2°C/s, 85 to 25°C at -0.3°C/s, and a 4°C hold. .. The heteroduplexes were processed using the Surveyor Mutation Detection Kit (Transgenomic, Omaha, NE, USA) and run on a 2% agarose gel to detect mutation efficiency.

Concentration Assay:

Article Title: In silico guided reconstruction and analysis of ICAM-1-binding var genes from Plasmodium falciparum
Article Snippet: .. Each reaction comprised 10 × LA PCR Buffer II, magnesium chloride (MgCl2 ) at 2.5 mM final concentration, dNTP at 1 mM final concentration, primers at 0.3 mM final concentration each, 2.5 units of TaKaRa LA Taq ®, 2 µl template and sterile water up to 50 µl final volume. .. The DBLα tag was amplified from parasite cDNA using the primers DBLαAF′ and DBLαBR .

BAC Assay:

Article Title: Molecular heterogeneity in the novel fusion gene APIP-FGFR2: Diversity of genomic breakpoints in gastric cancer with high-level amplifications at 11p13 and 10q26
Article Snippet: .. For the long-distance PCR, each reaction mixture (50 µl) contained 1 ng BAC DNA, 10 pmol of each primer, 8 µl of dNTP mixture (2.5 mM each), 5 µl LA PCR Buffer II and 2.5 U of Takara LA Taq HS (Takara Bio, Inc., Otsu, Japan). .. Reaction conditions were as follows: denaturation at 94°C for 1 min, followed by 30 cycles of denaturation at 98°C for 20 sec, annealing and extension at 68°C for 10 min with 15-sec increments per cycle, and a final extension at 72°C for 10 min. L4 contained exons 2–5 of APIP (nucleotides 118, 330-128, 595 in RP11-412L22), and L1 contained exons 11–13 of FGFR2 (nucleotides 27, 769-38, 427 in RP11-62L18).

Gel Extraction:

Article Title: Analysis of Chromosomal Numbers, Mitochondrial Genome, and Full-Length Transcriptome of Onychostoma brevibarba
Article Snippet: The amplifications were performed in a 25 μL reaction volume containing 1× LA PCR buffer II (Mg2+ ), 1.25 mM of dNTPs, 0.5 mM of each primer, 1.25 Unit of LA Taq polymerase (Takara, Dalian, China), and approximately 100 ng of template genomic DNA. .. Subsequently, the targeted fragments were purified using a gel extraction kit (Sangon, Shanghai, China) and directly sequenced by the Sangon Biotechnology Company (Sangon, Shanghai, China).

Fluorescence In Situ Hybridization:

Article Title: Molecular heterogeneity in the novel fusion gene APIP-FGFR2: Diversity of genomic breakpoints in gastric cancer with high-level amplifications at 11p13 and 10q26
Article Snippet: Paragraph title: Fluorescence in situ hybridization (FISH) analysis ... For the long-distance PCR, each reaction mixture (50 µl) contained 1 ng BAC DNA, 10 pmol of each primer, 8 µl of dNTP mixture (2.5 mM each), 5 µl LA PCR Buffer II and 2.5 U of Takara LA Taq HS (Takara Bio, Inc., Otsu, Japan).

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    TaKaRa la pcr buffer ii
    La Pcr Buffer Ii, supplied by TaKaRa, used in various techniques. Bioz Stars score: 90/100, based on 573 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/la pcr buffer ii/product/TaKaRa
    Average 90 stars, based on 573 article reviews
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    la pcr buffer ii - by Bioz Stars, 2020-01
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    80
    TaKaRa x la pcr buffer ii
    DREAM <t>PCR</t> and Déjà vu PCR makes use of what we have termed a <t>“DNA</t> diode” where enzymes that specifically digest 5 th and 6 th bases respectively are leveraged to ensure complex serial amplification steps can be performed contamination free without physical isolation of lab equipment. Both enzymes are heat inactivated and do not show activity post PCR. Any hmeC products cannot contaminate the Nextera reaction setup as AbaSI is present to selectively digest hmeC-DNA while leaving the target meC DNA intact. Likewise, any Nextera DNA contaminating the LR-PCR setup will be digested by MspJI since it that targets both forms of methylation.
    X La Pcr Buffer Ii, supplied by TaKaRa, used in various techniques. Bioz Stars score: 80/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/x la pcr buffer ii/product/TaKaRa
    Average 80 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    x la pcr buffer ii - by Bioz Stars, 2020-01
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      Buy from Supplier

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    DREAM PCR and Déjà vu PCR makes use of what we have termed a “DNA diode” where enzymes that specifically digest 5 th and 6 th bases respectively are leveraged to ensure complex serial amplification steps can be performed contamination free without physical isolation of lab equipment. Both enzymes are heat inactivated and do not show activity post PCR. Any hmeC products cannot contaminate the Nextera reaction setup as AbaSI is present to selectively digest hmeC-DNA while leaving the target meC DNA intact. Likewise, any Nextera DNA contaminating the LR-PCR setup will be digested by MspJI since it that targets both forms of methylation.

    Journal: PLoS ONE

    Article Title: Expanded Genetic Codes in Next Generation Sequencing Enable Decontamination and Mitochondrial Enrichment

    doi: 10.1371/journal.pone.0096492

    Figure Lengend Snippet: DREAM PCR and Déjà vu PCR makes use of what we have termed a “DNA diode” where enzymes that specifically digest 5 th and 6 th bases respectively are leveraged to ensure complex serial amplification steps can be performed contamination free without physical isolation of lab equipment. Both enzymes are heat inactivated and do not show activity post PCR. Any hmeC products cannot contaminate the Nextera reaction setup as AbaSI is present to selectively digest hmeC-DNA while leaving the target meC DNA intact. Likewise, any Nextera DNA contaminating the LR-PCR setup will be digested by MspJI since it that targets both forms of methylation.

    Article Snippet: Reaction setup included 1.5 ul of DNA, 5.0 ul of 10 X LA PCR Buffer II, 0.5 ul TaKaRa LA Taq DNA polymerase, 10.65 ul ddH20, and 0.125 ul (50 uM) of each primer with 8.0 ul dNTP mixture (2.5 mM each dNTP where a ratio of 87.5∶12.5 dCTP:5me-dCTP).

    Techniques: Polymerase Chain Reaction, Amplification, Isolation, Activity Assay, Methylation

    Deleted Mitochondrial DNA hyper-amplifies with LR-PCR. Observed vs Expected coverage of two unique haplogroup mtDNA samples pooled prior to LR-PCR amplification. One 4.5 kb Kearns-Sayre homozygous deleted mtDNA (12.1 kb, KSS mtDNA) sample is mixed with a known wild type mtDNA (16.6 kb, NA12878 mtDNA) sample with a different haplogroup. The KSS mtDNA sample has a unique haplogroup that creates heteroplasmies at expected loci when mixed with a full length mtDNA control. After sequencing the mixtures to 10,000× mean coverage on an Illumina MiSeq V2 system, allele frequencies are measured across a barcoded dilution series where the deleted sample alleles are expected to be seen at 5%,10%,15%,25%,50%,75% of the reads. Plotted is the expected coverage of the KSS mtDNA alleles versus the observed ratio (Y-Axis) of the control mtDNA alleles. This is measured by mapping reads with Bowtie and counting allele frequencies at the haplogroup specific loci. This result is expected in that a multiplexed PCR containing 12.1 kb and16.6 kb molecules will selectively amplify the smaller template. The selective amplification was still observed despite 15 minute extension times applied in PCR. This also highlights the pronounced sensitivity for detecting large deletions in mtDNA samples using LR-PCR.

    Journal: PLoS ONE

    Article Title: Expanded Genetic Codes in Next Generation Sequencing Enable Decontamination and Mitochondrial Enrichment

    doi: 10.1371/journal.pone.0096492

    Figure Lengend Snippet: Deleted Mitochondrial DNA hyper-amplifies with LR-PCR. Observed vs Expected coverage of two unique haplogroup mtDNA samples pooled prior to LR-PCR amplification. One 4.5 kb Kearns-Sayre homozygous deleted mtDNA (12.1 kb, KSS mtDNA) sample is mixed with a known wild type mtDNA (16.6 kb, NA12878 mtDNA) sample with a different haplogroup. The KSS mtDNA sample has a unique haplogroup that creates heteroplasmies at expected loci when mixed with a full length mtDNA control. After sequencing the mixtures to 10,000× mean coverage on an Illumina MiSeq V2 system, allele frequencies are measured across a barcoded dilution series where the deleted sample alleles are expected to be seen at 5%,10%,15%,25%,50%,75% of the reads. Plotted is the expected coverage of the KSS mtDNA alleles versus the observed ratio (Y-Axis) of the control mtDNA alleles. This is measured by mapping reads with Bowtie and counting allele frequencies at the haplogroup specific loci. This result is expected in that a multiplexed PCR containing 12.1 kb and16.6 kb molecules will selectively amplify the smaller template. The selective amplification was still observed despite 15 minute extension times applied in PCR. This also highlights the pronounced sensitivity for detecting large deletions in mtDNA samples using LR-PCR.

    Article Snippet: Reaction setup included 1.5 ul of DNA, 5.0 ul of 10 X LA PCR Buffer II, 0.5 ul TaKaRa LA Taq DNA polymerase, 10.65 ul ddH20, and 0.125 ul (50 uM) of each primer with 8.0 ul dNTP mixture (2.5 mM each dNTP where a ratio of 87.5∶12.5 dCTP:5me-dCTP).

    Techniques: Polymerase Chain Reaction, Amplification, Sequencing

    Decontamination effectiveness. To measure decontamination potential we mixed equimolar 5me-dCTP amplified mtDNA into non-methylated Target mtDNA. Methylated and non-methylated DNA were from mtDNA haplogroups differing in 8 loci. Each haplogroup mtDNA sample was barcoded with unique DNA barcodes prior to pooling, decontamination and amplification. Complete decontamination was measured via sequencing the mixed libraries to 10,000× coverage and measuring heteroplasmy levels with and without MspJI decontamination. MspJI digestion removed 100% of expected heteroplasmy contaminants(red) suggesting it can decontaminate up to equimolar contamination events. Undigested pooled libraries were sequenced as a control (blue) and exhibited 35–65% heteroplasmy levels. These artificial heteroplasmies were produced by pooling a methylated mitochondrial Long Range PCR product from a different haplogroup into a non methylated product. This haplogroup is completely removed by the decontamination methods described.

    Journal: PLoS ONE

    Article Title: Expanded Genetic Codes in Next Generation Sequencing Enable Decontamination and Mitochondrial Enrichment

    doi: 10.1371/journal.pone.0096492

    Figure Lengend Snippet: Decontamination effectiveness. To measure decontamination potential we mixed equimolar 5me-dCTP amplified mtDNA into non-methylated Target mtDNA. Methylated and non-methylated DNA were from mtDNA haplogroups differing in 8 loci. Each haplogroup mtDNA sample was barcoded with unique DNA barcodes prior to pooling, decontamination and amplification. Complete decontamination was measured via sequencing the mixed libraries to 10,000× coverage and measuring heteroplasmy levels with and without MspJI decontamination. MspJI digestion removed 100% of expected heteroplasmy contaminants(red) suggesting it can decontaminate up to equimolar contamination events. Undigested pooled libraries were sequenced as a control (blue) and exhibited 35–65% heteroplasmy levels. These artificial heteroplasmies were produced by pooling a methylated mitochondrial Long Range PCR product from a different haplogroup into a non methylated product. This haplogroup is completely removed by the decontamination methods described.

    Article Snippet: Reaction setup included 1.5 ul of DNA, 5.0 ul of 10 X LA PCR Buffer II, 0.5 ul TaKaRa LA Taq DNA polymerase, 10.65 ul ddH20, and 0.125 ul (50 uM) of each primer with 8.0 ul dNTP mixture (2.5 mM each dNTP where a ratio of 87.5∶12.5 dCTP:5me-dCTP).

    Techniques: Amplification, Methylation, Sequencing, Produced, Polymerase Chain Reaction

    Quantitative PCR of digested and undigested Déjà vu libraries. 120 minute digestion of AbaSI at 25°C on methylated DNA and hydroxymethylated DNA. A 100 fold reduction in background hydroxymethylated DNA is obtained with a 2 hr 25°C digestion with 0.3Units of Enzyme.

    Journal: PLoS ONE

    Article Title: Expanded Genetic Codes in Next Generation Sequencing Enable Decontamination and Mitochondrial Enrichment

    doi: 10.1371/journal.pone.0096492

    Figure Lengend Snippet: Quantitative PCR of digested and undigested Déjà vu libraries. 120 minute digestion of AbaSI at 25°C on methylated DNA and hydroxymethylated DNA. A 100 fold reduction in background hydroxymethylated DNA is obtained with a 2 hr 25°C digestion with 0.3Units of Enzyme.

    Article Snippet: Reaction setup included 1.5 ul of DNA, 5.0 ul of 10 X LA PCR Buffer II, 0.5 ul TaKaRa LA Taq DNA polymerase, 10.65 ul ddH20, and 0.125 ul (50 uM) of each primer with 8.0 ul dNTP mixture (2.5 mM each dNTP where a ratio of 87.5∶12.5 dCTP:5me-dCTP).

    Techniques: Real-time Polymerase Chain Reaction, Methylation