pcr  (New England Biolabs)


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    Structured Review

    New England Biolabs pcr
    Pcr, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcr/product/New England Biolabs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pcr - by Bioz Stars, 2021-06
    86/100 stars

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    Related Articles

    Mutagenesis:

    Article Title: Clamping, bending, and twisting inter-domain motions in the misfold-recognising portion of UDP-glucose:glycoprotein glucosyl-transferase
    Article Snippet: The pellets were resuspended, and the DNA purified using a Maxiprep kit (Qiagen), following the recommended protocol to obtain 3 mL of Ct UGGTA742C:Litmus28i plasmid DNA at 500 ng/µL. .. To obtain the double mutant Ct UGGTS180C/A742C , the second mutation S180C was introduced starting from the gene of Ct UGGTA742C in Litmus28i as follows: 12.5 µl of Q5® Hot Start High-Fidelity 2X Master Mix (New England Biolabs) were added to 1.25 µl of each forward and reverse primers (primers S180C_F and S180C_R, see ) at 10 µM, 1 µl of Ct UGGTA742C :Litmus28i DNA at 1 ng/µL and 9 µl of nuclease-free water, obtaining a 25 µl final volume. .. PCR protocol: step 1: 98 °C for 30 seconds; step 2: 98 °C for 10 seconds; step 3: 68 °C for 30 seconds; step 4: 72 °C for 135 seconds; steps 2-4 were repeated 25 times.

    Immunoprecipitation:

    Article Title: Single-cell profiling of histone modifications in the mouse brain
    Article Snippet: Briefly, chromatin was sonicated using the covaries ME220 with settings 75 PIP, 5% duty cycle, 200 cycles/burst for 2 minutes (for 1-3 milion cells). .. The immunoprecipitation was performed using 2 ug of H3K27ac antibody (Abcam, Ab177178) and 20 ul of protein A dynabeads (Thermo Fisher, 007613560), with 0.75 ul of in-house produced Tn5 used for tagmentation and 15-16 cycles of final PCR amplification (NEBNext High Fidelity 2x PCR mastermix, M0541L). ..

    Produced:

    Article Title: Single-cell profiling of histone modifications in the mouse brain
    Article Snippet: Briefly, chromatin was sonicated using the covaries ME220 with settings 75 PIP, 5% duty cycle, 200 cycles/burst for 2 minutes (for 1-3 milion cells). .. The immunoprecipitation was performed using 2 ug of H3K27ac antibody (Abcam, Ab177178) and 20 ul of protein A dynabeads (Thermo Fisher, 007613560), with 0.75 ul of in-house produced Tn5 used for tagmentation and 15-16 cycles of final PCR amplification (NEBNext High Fidelity 2x PCR mastermix, M0541L). ..

    Polymerase Chain Reaction:

    Article Title: Single-cell profiling of histone modifications in the mouse brain
    Article Snippet: Briefly, chromatin was sonicated using the covaries ME220 with settings 75 PIP, 5% duty cycle, 200 cycles/burst for 2 minutes (for 1-3 milion cells). .. The immunoprecipitation was performed using 2 ug of H3K27ac antibody (Abcam, Ab177178) and 20 ul of protein A dynabeads (Thermo Fisher, 007613560), with 0.75 ul of in-house produced Tn5 used for tagmentation and 15-16 cycles of final PCR amplification (NEBNext High Fidelity 2x PCR mastermix, M0541L). ..

    Article Title: FGFR1OP tagSNP but Not CCR6 Polymorphisms Are Associated with Vogt-Koyanagi-Harada Syndrome in Chinese Han
    Article Snippet: These SNPs were genotyped by PCR-restriction fragment length polymorphism analysis. .. PCR products of rs3093024, rs6902119, rs3093023, rs968334 and rs2301436 polymorphisms were respectively digested with 2 U of BtsI (New England Biolabs, Inc., Ontario, Canada), PvuII (New England Biolabs, Inc., Ontario, Canada), TspRI (New England Biolabs, Inc., Ontario, Canada), and MspI (New England Biolabs, Inc., Ontario, Canada) restriction enzymes ( ) in a 10 µl reaction volume overnight. .. Digestion products were visualized on a 3.5% agarose gel and stained with GoldView (SBS Genetech, Beijing, China)( ).

    Amplification:

    Article Title: Single-cell profiling of histone modifications in the mouse brain
    Article Snippet: Briefly, chromatin was sonicated using the covaries ME220 with settings 75 PIP, 5% duty cycle, 200 cycles/burst for 2 minutes (for 1-3 milion cells). .. The immunoprecipitation was performed using 2 ug of H3K27ac antibody (Abcam, Ab177178) and 20 ul of protein A dynabeads (Thermo Fisher, 007613560), with 0.75 ul of in-house produced Tn5 used for tagmentation and 15-16 cycles of final PCR amplification (NEBNext High Fidelity 2x PCR mastermix, M0541L). ..

    Article Title: Histone Deacetylases SRT1 and SRT2 Interact with ENAP1 to Mediate Ethylene-Induced Transcriptional Repression [OPEN]
    Article Snippet: .. To construct vectors for yeast two-hybrid analysis, the coding sequences (CDSs) of HDAC s, EIN2-C , ENAP1 , and EIN3 were amplified using the Phusion High-Fidelity DNA Polymerase (NEB). ..

    Construct:

    Article Title: Histone Deacetylases SRT1 and SRT2 Interact with ENAP1 to Mediate Ethylene-Induced Transcriptional Repression [OPEN]
    Article Snippet: .. To construct vectors for yeast two-hybrid analysis, the coding sequences (CDSs) of HDAC s, EIN2-C , ENAP1 , and EIN3 were amplified using the Phusion High-Fidelity DNA Polymerase (NEB). ..

    Injection:

    Article Title: The C. elegans neural editome reveals an ADAR target mRNA required for proper chemotaxis
    Article Snippet: Transgenic worm line generation Transgenic worm lines were generated by microinjection into the gonads of young adult N2 worms. .. The injection mix used for generating transgenic worms contained the following: 10 ng/μl of the transgene of interest, 20 ng/μl of the dominant marker, and 70 ng/μl of 1 kb DNA ladder (NEB, Ipswich, MA). .. Transgenic strains were maintained by passaging only worms with the dominant marker.

    Transgenic Assay:

    Article Title: The C. elegans neural editome reveals an ADAR target mRNA required for proper chemotaxis
    Article Snippet: Transgenic worm line generation Transgenic worm lines were generated by microinjection into the gonads of young adult N2 worms. .. The injection mix used for generating transgenic worms contained the following: 10 ng/μl of the transgene of interest, 20 ng/μl of the dominant marker, and 70 ng/μl of 1 kb DNA ladder (NEB, Ipswich, MA). .. Transgenic strains were maintained by passaging only worms with the dominant marker.

    Marker:

    Article Title: The C. elegans neural editome reveals an ADAR target mRNA required for proper chemotaxis
    Article Snippet: Transgenic worm line generation Transgenic worm lines were generated by microinjection into the gonads of young adult N2 worms. .. The injection mix used for generating transgenic worms contained the following: 10 ng/μl of the transgene of interest, 20 ng/μl of the dominant marker, and 70 ng/μl of 1 kb DNA ladder (NEB, Ipswich, MA). .. Transgenic strains were maintained by passaging only worms with the dominant marker.

    Cell Culture:

    Article Title: Serotype-specific interactions among functional domains of dengue virus 2 nonstructural proteins (NS) 5 and NS3 are crucial for viral RNA replication
    Article Snippet: .. The other samples (330–800 ng) from WT DENV2 RNA from cultured BHK-21 cells and DENV2 chimeras containing the DENV4 POL (264–900 aa), DENV4 MTase, and DENV4 NS3hel + NS5 domains were subjected to NEBNext rRNA depletion (for mammalian cells). ..

    Quantitative RT-PCR:

    Article Title: Novel small RNAs expressed by Bartonella bacilliformis under multiple conditions reveal potential mechanisms for persistence in the sand fly vector and human host
    Article Snippet: Membranes were washed 3 times for 15 min at 67°C in 1X Hybridization Stringency Wash Buffer (Thermo Fisher), developed, and imaged with a ChemiDoc XRS+ system (Bio-Rad). .. qRT-PCR qRT-PCR was done on cDNA synthesized from 16 ng B. bacilliformis total RNA (for each 25 µl reaction) collected from various conditions using the Luna Universal One-Step RT-qPCR kit (New England BioLabs; Ipswich, MA) according to the manufacturer. .. B. bacilliformis total RNA was serially diluted and used as a standard curve, while primers targeting the rpoD housekeeping gene were used for normalization of gene expression between conditions. qRT-PCR was performed on a CFX Connect Real-Time System (Bio-Rad). cDNA from sRNAs of interest was analyzed for copy number, then divided by the copy number from the rpoD gene to achieve the sRNA transcripts / rpoD transcript values.

    Synthesized:

    Article Title: Novel small RNAs expressed by Bartonella bacilliformis under multiple conditions reveal potential mechanisms for persistence in the sand fly vector and human host
    Article Snippet: Membranes were washed 3 times for 15 min at 67°C in 1X Hybridization Stringency Wash Buffer (Thermo Fisher), developed, and imaged with a ChemiDoc XRS+ system (Bio-Rad). .. qRT-PCR qRT-PCR was done on cDNA synthesized from 16 ng B. bacilliformis total RNA (for each 25 µl reaction) collected from various conditions using the Luna Universal One-Step RT-qPCR kit (New England BioLabs; Ipswich, MA) according to the manufacturer. .. B. bacilliformis total RNA was serially diluted and used as a standard curve, while primers targeting the rpoD housekeeping gene were used for normalization of gene expression between conditions. qRT-PCR was performed on a CFX Connect Real-Time System (Bio-Rad). cDNA from sRNAs of interest was analyzed for copy number, then divided by the copy number from the rpoD gene to achieve the sRNA transcripts / rpoD transcript values.

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    New England Biolabs monarch pcr and dna cleanup kit
    mRNA reporter design and in-cell and in-solution workflows with in-cell polysome validation. (A) Schematic for the 3’ UTR-barcoded mRNA reporter used to screen mRNA performance in a pooled format. The constant regions and barcode, which flank a variable 3’ UTR, were instrumental for amplifying and identifying hundreds of constructs simultaneously in each of the pooled experiments that comprise PERSIST-seq. The <t>DNA</t> templates for full-length mRNAs were synthesized on the Codex platform and amplified in a pooled <t>PCR</t> using primers complementary to the constant region (T7 promoter) preceding the variable 5’ UTR, and to the ‘constant3’ region following the variable 3’ UTR. (B) Summary of the workflow to progress from the individually synthesized DNA templates to the in vitro synthesized mRNA pool of 233 different constructs. We then use the same mRNA pool to screen mRNA performance in a three-pronged set of in-cell and in-solution expression and stability analyses. (C) Quality control of the 233-mRNA pool on a 1.2% formaldehyde (FA) gel stained with ethidium bromide (EtBr) after 3 hrs of in vitro transcription (IVT). The mRNA pool was analyzed before and after capping and polyadenylation. Pooled IVT is equally efficient with the starting template DNA pool with or without PCR-amplification of the DNA template pool. The three major bands corresponding to the three CDS types are indicated. The RiboRuler High Range RNA ladder (Thermo Fisher) is loaded for reference. (D) Polysome fractionation analysis of a transfected mRNA reporter. As an example, the distribution of an mRNA with short scrambled 5’ and 3’ UTRs 6 hrs after transfection into HEK293T cells was compared to the distribution of endogenous human ActB mRNA. RNA was extracted from fractions and quantified by qPCR with a RNA spike-in for normalization. Values are plotted as mRNA normalized per fraction. (E) In-solution RNA degradation strategy of barcoded mRNAs containing CDS variants with hHBB 5’ and 3’ UTRs. The differential degradation of CDS variants depends on their individual CDS structures. mRNA pools are degraded in solution by nucleophilic attack (red circle). After degradation, RT-PCR is performed to selectively amplify mRNAs that remain intact along their full length. Then, the barcode regions of these full-length mRNAs are PCR-amplified, adaptor-ligated, and prepared for Illumina sequencing.
    Monarch Pcr And Dna Cleanup Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monarch pcr and dna cleanup kit/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    99
    New England Biolabs pcr amplified
    5hmC favours co-transcriptional R-loop formation. (A) Native or modified dCTPs were incorporated upon <t>PCR</t> amplification into <t>DNA</t> fragments with sequences from the transcription termination region of the β- actin gene (β- actin P1 and β- actin P2) or the APOE gene. (B) Incorporation of dCTP variants confirmed by immunoblotting using specific antibodies against 5mC, 5hmC and double-stranded DNA (dsDNA). (C) R-loops formed upon in vitro transcription reactions were detected by immunoblotting using the S9.6 antibody. RNase H-treated in vitro transcription reaction products (RH+) serve as negative controls. All data are representative of seven independent experiments with similar results. (D) S9.6 immunoblots were quantified and the R-loop levels normalized against the levels detected in the reaction products of DNA templates containing native C. Data represent the mean and standard deviation (SD) from seven independent experiments. *p
    Pcr Amplified, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcr amplified/product/New England Biolabs
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    86
    New England Biolabs polymerase chain reaction pcr assays
    Successful <t>PCRs</t> on genomic DNA extracted from one or both wings by using the custom method. (A) Quantification of successful PCRs on the alpha-tubulin 1 upstream regulatory sequence from five replicates with ten adults each using genomic DNA extracted from one or both wings as template. Significantly more successful PCRs could be performed when only one wing was digested. Error bars represent standard deviation. (B) Exemplary agarose gel electrophoresis images showing the <t>PCR-based</t> amplification of the alpha-tubulin 1 upstream regulatory sequence (ATub’) for ten individual adults using DNA extracted from either one or both wings. The estimated PCR product size is 616 bp. The bright DNA marker bands (top to bottom) represent 3,000 / 1,000 / 500 bp. In the negative control (–), double-distilled H 2 O was used as template and in the positive control (+), DNA extracted from the whole body was used as template.
    Polymerase Chain Reaction Pcr Assays, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/polymerase chain reaction pcr assays/product/New England Biolabs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    Removal of excess primers and unincorporated dNTPs from PCR products is recommended prior to downstream applications and analysis The Exo CIP Rapid PCR Cleanup Kit serves this purpose by utilizing
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    mRNA reporter design and in-cell and in-solution workflows with in-cell polysome validation. (A) Schematic for the 3’ UTR-barcoded mRNA reporter used to screen mRNA performance in a pooled format. The constant regions and barcode, which flank a variable 3’ UTR, were instrumental for amplifying and identifying hundreds of constructs simultaneously in each of the pooled experiments that comprise PERSIST-seq. The DNA templates for full-length mRNAs were synthesized on the Codex platform and amplified in a pooled PCR using primers complementary to the constant region (T7 promoter) preceding the variable 5’ UTR, and to the ‘constant3’ region following the variable 3’ UTR. (B) Summary of the workflow to progress from the individually synthesized DNA templates to the in vitro synthesized mRNA pool of 233 different constructs. We then use the same mRNA pool to screen mRNA performance in a three-pronged set of in-cell and in-solution expression and stability analyses. (C) Quality control of the 233-mRNA pool on a 1.2% formaldehyde (FA) gel stained with ethidium bromide (EtBr) after 3 hrs of in vitro transcription (IVT). The mRNA pool was analyzed before and after capping and polyadenylation. Pooled IVT is equally efficient with the starting template DNA pool with or without PCR-amplification of the DNA template pool. The three major bands corresponding to the three CDS types are indicated. The RiboRuler High Range RNA ladder (Thermo Fisher) is loaded for reference. (D) Polysome fractionation analysis of a transfected mRNA reporter. As an example, the distribution of an mRNA with short scrambled 5’ and 3’ UTRs 6 hrs after transfection into HEK293T cells was compared to the distribution of endogenous human ActB mRNA. RNA was extracted from fractions and quantified by qPCR with a RNA spike-in for normalization. Values are plotted as mRNA normalized per fraction. (E) In-solution RNA degradation strategy of barcoded mRNAs containing CDS variants with hHBB 5’ and 3’ UTRs. The differential degradation of CDS variants depends on their individual CDS structures. mRNA pools are degraded in solution by nucleophilic attack (red circle). After degradation, RT-PCR is performed to selectively amplify mRNAs that remain intact along their full length. Then, the barcode regions of these full-length mRNAs are PCR-amplified, adaptor-ligated, and prepared for Illumina sequencing.

    Journal: bioRxiv

    Article Title: Combinatorial optimization of mRNA structure, stability, and translation for RNA-based therapeutics

    doi: 10.1101/2021.03.29.437587

    Figure Lengend Snippet: mRNA reporter design and in-cell and in-solution workflows with in-cell polysome validation. (A) Schematic for the 3’ UTR-barcoded mRNA reporter used to screen mRNA performance in a pooled format. The constant regions and barcode, which flank a variable 3’ UTR, were instrumental for amplifying and identifying hundreds of constructs simultaneously in each of the pooled experiments that comprise PERSIST-seq. The DNA templates for full-length mRNAs were synthesized on the Codex platform and amplified in a pooled PCR using primers complementary to the constant region (T7 promoter) preceding the variable 5’ UTR, and to the ‘constant3’ region following the variable 3’ UTR. (B) Summary of the workflow to progress from the individually synthesized DNA templates to the in vitro synthesized mRNA pool of 233 different constructs. We then use the same mRNA pool to screen mRNA performance in a three-pronged set of in-cell and in-solution expression and stability analyses. (C) Quality control of the 233-mRNA pool on a 1.2% formaldehyde (FA) gel stained with ethidium bromide (EtBr) after 3 hrs of in vitro transcription (IVT). The mRNA pool was analyzed before and after capping and polyadenylation. Pooled IVT is equally efficient with the starting template DNA pool with or without PCR-amplification of the DNA template pool. The three major bands corresponding to the three CDS types are indicated. The RiboRuler High Range RNA ladder (Thermo Fisher) is loaded for reference. (D) Polysome fractionation analysis of a transfected mRNA reporter. As an example, the distribution of an mRNA with short scrambled 5’ and 3’ UTRs 6 hrs after transfection into HEK293T cells was compared to the distribution of endogenous human ActB mRNA. RNA was extracted from fractions and quantified by qPCR with a RNA spike-in for normalization. Values are plotted as mRNA normalized per fraction. (E) In-solution RNA degradation strategy of barcoded mRNAs containing CDS variants with hHBB 5’ and 3’ UTRs. The differential degradation of CDS variants depends on their individual CDS structures. mRNA pools are degraded in solution by nucleophilic attack (red circle). After degradation, RT-PCR is performed to selectively amplify mRNAs that remain intact along their full length. Then, the barcode regions of these full-length mRNAs are PCR-amplified, adaptor-ligated, and prepared for Illumina sequencing.

    Article Snippet: PCR reactions were purified with Monarch PCR & DNA Cleanup Kit (NEB, T1030L).

    Techniques: Construct, Synthesized, Amplification, Polymerase Chain Reaction, In Vitro, Expressing, Staining, Fractionation, Transfection, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Sequencing

    Mutations were detected in FKH domains of FOXP3 transcripts in HCC. A , representative sequencing chromatograms of point mutations. B , representative sequencing chromatograms of the complicated mutation status. Upper left panel , FKH sequences in mRNA. Upper right panel , sequences of the corresponding regions in genomic DNA. Lower panel , sequences of individual TA clones which were generated to examine individual sequences in multiple-mutation bearing PCR products. C , representative immunohistochemical results of FOXP3-positive lymphocyte distribution in tumors and corresponding nontumorous tissues. Black arrow , FOXP3-positive lymphocytes.

    Journal: The Journal of Biological Chemistry

    Article Title: The FKH domain in FOXP3 mRNA frequently contains mutations in hepatocellular carcinoma that influence the subcellular localization and functions of FOXP3

    doi: 10.1074/jbc.RA120.012518

    Figure Lengend Snippet: Mutations were detected in FKH domains of FOXP3 transcripts in HCC. A , representative sequencing chromatograms of point mutations. B , representative sequencing chromatograms of the complicated mutation status. Upper left panel , FKH sequences in mRNA. Upper right panel , sequences of the corresponding regions in genomic DNA. Lower panel , sequences of individual TA clones which were generated to examine individual sequences in multiple-mutation bearing PCR products. C , representative immunohistochemical results of FOXP3-positive lymphocyte distribution in tumors and corresponding nontumorous tissues. Black arrow , FOXP3-positive lymphocytes.

    Article Snippet: The 1.2-kb PCR products were purified with Monarch PCR & DNA Cleanup Kit (New England Biolabs) and sent to BGI (Shenzhen, China) for sequencing with primer 5′-GTAGCCATGGAAACAGCACA-3′.

    Techniques: Sequencing, Mutagenesis, Clone Assay, Generated, Polymerase Chain Reaction, Immunohistochemistry

    5hmC favours co-transcriptional R-loop formation. (A) Native or modified dCTPs were incorporated upon PCR amplification into DNA fragments with sequences from the transcription termination region of the β- actin gene (β- actin P1 and β- actin P2) or the APOE gene. (B) Incorporation of dCTP variants confirmed by immunoblotting using specific antibodies against 5mC, 5hmC and double-stranded DNA (dsDNA). (C) R-loops formed upon in vitro transcription reactions were detected by immunoblotting using the S9.6 antibody. RNase H-treated in vitro transcription reaction products (RH+) serve as negative controls. All data are representative of seven independent experiments with similar results. (D) S9.6 immunoblots were quantified and the R-loop levels normalized against the levels detected in the reaction products of DNA templates containing native C. Data represent the mean and standard deviation (SD) from seven independent experiments. *p

    Journal: bioRxiv

    Article Title: Epigenetic reprogramming by TET enzymes impacts co-transcriptional R-loops

    doi: 10.1101/2021.04.26.441414

    Figure Lengend Snippet: 5hmC favours co-transcriptional R-loop formation. (A) Native or modified dCTPs were incorporated upon PCR amplification into DNA fragments with sequences from the transcription termination region of the β- actin gene (β- actin P1 and β- actin P2) or the APOE gene. (B) Incorporation of dCTP variants confirmed by immunoblotting using specific antibodies against 5mC, 5hmC and double-stranded DNA (dsDNA). (C) R-loops formed upon in vitro transcription reactions were detected by immunoblotting using the S9.6 antibody. RNase H-treated in vitro transcription reaction products (RH+) serve as negative controls. All data are representative of seven independent experiments with similar results. (D) S9.6 immunoblots were quantified and the R-loop levels normalized against the levels detected in the reaction products of DNA templates containing native C. Data represent the mean and standard deviation (SD) from seven independent experiments. *p

    Article Snippet: g-blocks PCR Designed g-blocks were ordered from IDT , and PCR-amplified using Phusion High-Fidelity DNA Polymerase (M0530S, NEB), according to manufacturer’s instructions.

    Techniques: Modification, Polymerase Chain Reaction, Amplification, In Vitro, Western Blot, Standard Deviation

    Successful PCRs on genomic DNA extracted from one or both wings by using the custom method. (A) Quantification of successful PCRs on the alpha-tubulin 1 upstream regulatory sequence from five replicates with ten adults each using genomic DNA extracted from one or both wings as template. Significantly more successful PCRs could be performed when only one wing was digested. Error bars represent standard deviation. (B) Exemplary agarose gel electrophoresis images showing the PCR-based amplification of the alpha-tubulin 1 upstream regulatory sequence (ATub’) for ten individual adults using DNA extracted from either one or both wings. The estimated PCR product size is 616 bp. The bright DNA marker bands (top to bottom) represent 3,000 / 1,000 / 500 bp. In the negative control (–), double-distilled H 2 O was used as template and in the positive control (+), DNA extracted from the whole body was used as template.

    Journal: PLoS ONE

    Article Title: Non-lethal genotyping of Tribolium castaneum adults using genomic DNA extracted from wing tissue

    doi: 10.1371/journal.pone.0182564

    Figure Lengend Snippet: Successful PCRs on genomic DNA extracted from one or both wings by using the custom method. (A) Quantification of successful PCRs on the alpha-tubulin 1 upstream regulatory sequence from five replicates with ten adults each using genomic DNA extracted from one or both wings as template. Significantly more successful PCRs could be performed when only one wing was digested. Error bars represent standard deviation. (B) Exemplary agarose gel electrophoresis images showing the PCR-based amplification of the alpha-tubulin 1 upstream regulatory sequence (ATub’) for ten individual adults using DNA extracted from either one or both wings. The estimated PCR product size is 616 bp. The bright DNA marker bands (top to bottom) represent 3,000 / 1,000 / 500 bp. In the negative control (–), double-distilled H 2 O was used as template and in the positive control (+), DNA extracted from the whole body was used as template.

    Article Snippet: Polymerase chain reaction (PCR) assays All PCRs were performed with the Phusion polymerase (M0530L, New England BioLabs) in 20 μl reactions.

    Techniques: Sequencing, Standard Deviation, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Marker, Negative Control, Positive Control

    Sequencing of the SB and Prl white allele. (A) Exon / intron structure of the white gene (8,376 bp) with respective primer groups for extraction PCRs. (B) Agarose gel electrophoresis image showing the results for the extraction PCRs. Estimated PCR product size is 616 bp for the alpha-tubulin 1 upstream regulatory sequence control (ATub’), 4,208 bp for the exon 1–6 primer group and 4,295 bp for the exon 6–10 primer group. Bright DNA marker bands (top to bottom) represent 3,000 / 1,000 / 500 bp. In the negative control (–), double-distilled H 2 O was used as template. (C) Alignment of the SB and Prl white sequences. Vertical black bars represent sequence deviations, horizontal black lines show insertions / deletions. The red asterisks mark the two mutations that lead to amino acid exchanges (Y437V and G573S).

    Journal: PLoS ONE

    Article Title: Non-lethal genotyping of Tribolium castaneum adults using genomic DNA extracted from wing tissue

    doi: 10.1371/journal.pone.0182564

    Figure Lengend Snippet: Sequencing of the SB and Prl white allele. (A) Exon / intron structure of the white gene (8,376 bp) with respective primer groups for extraction PCRs. (B) Agarose gel electrophoresis image showing the results for the extraction PCRs. Estimated PCR product size is 616 bp for the alpha-tubulin 1 upstream regulatory sequence control (ATub’), 4,208 bp for the exon 1–6 primer group and 4,295 bp for the exon 6–10 primer group. Bright DNA marker bands (top to bottom) represent 3,000 / 1,000 / 500 bp. In the negative control (–), double-distilled H 2 O was used as template. (C) Alignment of the SB and Prl white sequences. Vertical black bars represent sequence deviations, horizontal black lines show insertions / deletions. The red asterisks mark the two mutations that lead to amino acid exchanges (Y437V and G573S).

    Article Snippet: Polymerase chain reaction (PCR) assays All PCRs were performed with the Phusion polymerase (M0530L, New England BioLabs) in 20 μl reactions.

    Techniques: Sequencing, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Marker, Negative Control