datp  (New England Biolabs)


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    Name:
    dATP Solution
    Description:
    dATP Solution 25 umol
    Catalog Number:
    n0440s
    Price:
    53
    Size:
    25 umol
    Category:
    Deoxynucleotides
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    Structured Review

    New England Biolabs datp
    dATP Solution
    dATP Solution 25 umol
    https://www.bioz.com/result/datp/product/New England Biolabs
    Average 97 stars, based on 161 article reviews
    Price from $9.99 to $1999.99
    datp - by Bioz Stars, 2020-01
    97/100 stars

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    1) Product Images from "High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection"

    Article Title: High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0018900

    Nucleotide sequences of integrated oligonucleotide fragments. Sequences of integrated oligonucleotide fragments with features common to all LIC-LC1 and LIC-LC2 vectors are shown. Double-stranded oligonucleotides were integrated at the restriction enzyme recognition sites indicated except for PmeI which is used to eliminate the 670-bp stuffer fragment prior to the LIC process. LIC-pPICZ-LC1/-LC2 vectors were generated by inserting AclI/SalI-restricted double-stranded oligonucleotides into BstBI/SalI-digested expression vector (cutting with AclI and BstBI creates compatible 5′ overhangs), resulting in a change of the BstBI sequence (TTCGAA to TTCGTT). The asterisk on the forward strand indicates the position of adenine (corresponding to thymine on the reverse strand) required for the generation of LIC 5′ overhangs in the presence of T4 DNA polymerase and dATP. The blue arrow indicates the TEV cleavage site suitable for the removal of the marker proteins IFP and 6xHis-tag.
    Figure Legend Snippet: Nucleotide sequences of integrated oligonucleotide fragments. Sequences of integrated oligonucleotide fragments with features common to all LIC-LC1 and LIC-LC2 vectors are shown. Double-stranded oligonucleotides were integrated at the restriction enzyme recognition sites indicated except for PmeI which is used to eliminate the 670-bp stuffer fragment prior to the LIC process. LIC-pPICZ-LC1/-LC2 vectors were generated by inserting AclI/SalI-restricted double-stranded oligonucleotides into BstBI/SalI-digested expression vector (cutting with AclI and BstBI creates compatible 5′ overhangs), resulting in a change of the BstBI sequence (TTCGAA to TTCGTT). The asterisk on the forward strand indicates the position of adenine (corresponding to thymine on the reverse strand) required for the generation of LIC 5′ overhangs in the presence of T4 DNA polymerase and dATP. The blue arrow indicates the TEV cleavage site suitable for the removal of the marker proteins IFP and 6xHis-tag.

    Techniques Used: Generated, Expressing, Plasmid Preparation, Sequencing, Marker

    Ligation-independent cloning using LIC-IFP-compatible expression vectors. LIC vectors (LIC-LC1 and LIC-LC2) are cleaved with PmeI restriction enzyme and the released stuffer fragment (670 bp) is removed. The cleaved vector is treated with T4 DNA polymerase in the presence of dATP, whereas the PCR product (amplified open reading frame) is treated in the presence of dTTP. The asterisks indicate the position of adenine (vector) or thymine (PCR product) required for the generation of LIC-complementary 5′ overhangs. After successful annealing and transformation into E. coli , host-internal ligases and DNA polymerases close the vector and fill in the gaps, caused by the two additional nucleotides (CC, coloured in blue) upstream of the start codon (ATG), which are required to retain the reading frame. For LIC with LC1 vectors, PCR-amplified open reading frames contain a double stop codon (TAATAG); for LIC with LC2 vectors, open reading frames must not contain a stop codon to allow expression of ProteinX-TEV-IFP-6xHis fusion proteins. To provide the thymine moiety on the forward strand for dTTP/T4 DNA polymerase treatment, additional three nucleotides (GGT) are added directly at the 3′-end of the PCR-amplified open reading frame.
    Figure Legend Snippet: Ligation-independent cloning using LIC-IFP-compatible expression vectors. LIC vectors (LIC-LC1 and LIC-LC2) are cleaved with PmeI restriction enzyme and the released stuffer fragment (670 bp) is removed. The cleaved vector is treated with T4 DNA polymerase in the presence of dATP, whereas the PCR product (amplified open reading frame) is treated in the presence of dTTP. The asterisks indicate the position of adenine (vector) or thymine (PCR product) required for the generation of LIC-complementary 5′ overhangs. After successful annealing and transformation into E. coli , host-internal ligases and DNA polymerases close the vector and fill in the gaps, caused by the two additional nucleotides (CC, coloured in blue) upstream of the start codon (ATG), which are required to retain the reading frame. For LIC with LC1 vectors, PCR-amplified open reading frames contain a double stop codon (TAATAG); for LIC with LC2 vectors, open reading frames must not contain a stop codon to allow expression of ProteinX-TEV-IFP-6xHis fusion proteins. To provide the thymine moiety on the forward strand for dTTP/T4 DNA polymerase treatment, additional three nucleotides (GGT) are added directly at the 3′-end of the PCR-amplified open reading frame.

    Techniques Used: Ligation, Clone Assay, Expressing, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Transformation Assay

    Related Articles

    Clone Assay:

    Article Title: High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection
    Article Snippet: Paragraph title: Linearization of LIC expression vectors for LIC cloning ... To generate 5′ LIC overhangs (15 and 16 nt, respectively) at both ends the purified vector backbone was treated for 30 min (22°C) with T4 DNA polymerase in the presence of dATP, using the following reaction setup: 0.2 pmol purified vector backbone, 2 µL 10× buffer 2 (NEB), 2 µL dATP (25 mM), 1 µL dithiothreitol (DTT, 100 mM), 2 µL 10× (10 mg/mL) bovine serum albumin (BSA; NEB), 10 U T4 DNA polymerase (NEB) in a volume of 20 µL (filled up with ddH2 O).

    Selection:

    Article Title: Passing experiences on to future generations: endocrine disruptors and transgenerational inheritance of epimutations in brain and sperm
    Article Snippet: Gap-filling and A-tailing were performed in a single step with 3’ - >  5’ exo- Klenow fragment (NEB, M0212) and an abundance of dATP (10 mM) in a dNTP (1 mM) mixture at 30 C for 20 minutes followed by 37 C for 20 minutes (NEB, N0446). .. The enzymes and dNTPs were removed and size selection was performed with a 2.0X concentration of Agencourt AMPure XP beads (Beckman Coulter, A63880) and eluted in 0.1X TE.

    Methylation:

    Article Title: Passing experiences on to future generations: endocrine disruptors and transgenerational inheritance of epimutations in brain and sperm
    Article Snippet: Gap-filling and A-tailing were performed in a single step with 3’ - >  5’ exo- Klenow fragment (NEB, M0212) and an abundance of dATP (10 mM) in a dNTP (1 mM) mixture at 30 C for 20 minutes followed by 37 C for 20 minutes (NEB, N0446). .. Adaptor ligation was performed using methylated adaptors (Index Primers Set 1 for sperm and 1–4 for brain, NEB, E7535) and TA/Blunt ligase master mix (NEB, M0367) for 15 minutes at room temperature.

    Ligation:

    Article Title: Passing experiences on to future generations: endocrine disruptors and transgenerational inheritance of epimutations in brain and sperm
    Article Snippet: Gap-filling and A-tailing were performed in a single step with 3’ - >  5’ exo- Klenow fragment (NEB, M0212) and an abundance of dATP (10 mM) in a dNTP (1 mM) mixture at 30 C for 20 minutes followed by 37 C for 20 minutes (NEB, N0446). .. Adaptor ligation was performed using methylated adaptors (Index Primers Set 1 for sperm and 1–4 for brain, NEB, E7535) and TA/Blunt ligase master mix (NEB, M0367) for 15 minutes at room temperature.

    Purification:

    Article Title: High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection
    Article Snippet: .. To generate 5′ LIC overhangs (15 and 16 nt, respectively) at both ends the purified vector backbone was treated for 30 min (22°C) with T4 DNA polymerase in the presence of dATP, using the following reaction setup: 0.2 pmol purified vector backbone, 2 µL 10× buffer 2 (NEB), 2 µL dATP (25 mM), 1 µL dithiothreitol (DTT, 100 mM), 2 µL 10× (10 mg/mL) bovine serum albumin (BSA; NEB), 10 U T4 DNA polymerase (NEB) in a volume of 20 µL (filled up with ddH2 O). .. The reaction mix was heat inactivated for 20 min at 75°C, followed by purification using the NucleoSpin Extract II kit (Macherey & Nagel) and elution with 20 µL elution buffer included in the kit.

    Concentration Assay:

    Article Title: Passing experiences on to future generations: endocrine disruptors and transgenerational inheritance of epimutations in brain and sperm
    Article Snippet: Gap-filling and A-tailing were performed in a single step with 3’ - >  5’ exo- Klenow fragment (NEB, M0212) and an abundance of dATP (10 mM) in a dNTP (1 mM) mixture at 30 C for 20 minutes followed by 37 C for 20 minutes (NEB, N0446). .. The enzymes and dNTPs were removed and size selection was performed with a 2.0X concentration of Agencourt AMPure XP beads (Beckman Coulter, A63880) and eluted in 0.1X TE.

    Expressing:

    Article Title: High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection
    Article Snippet: Paragraph title: Linearization of LIC expression vectors for LIC cloning ... To generate 5′ LIC overhangs (15 and 16 nt, respectively) at both ends the purified vector backbone was treated for 30 min (22°C) with T4 DNA polymerase in the presence of dATP, using the following reaction setup: 0.2 pmol purified vector backbone, 2 µL 10× buffer 2 (NEB), 2 µL dATP (25 mM), 1 µL dithiothreitol (DTT, 100 mM), 2 µL 10× (10 mg/mL) bovine serum albumin (BSA; NEB), 10 U T4 DNA polymerase (NEB) in a volume of 20 µL (filled up with ddH2 O).

    Modification:

    Article Title: Passing experiences on to future generations: endocrine disruptors and transgenerational inheritance of epimutations in brain and sperm
    Article Snippet: Library preparation was performed by modification of previously established protocols [ ] and the manufacturer’s protocols (NEB, E7535). .. Gap-filling and A-tailing were performed in a single step with 3’ - >  5’ exo- Klenow fragment (NEB, M0212) and an abundance of dATP (10 mM) in a dNTP (1 mM) mixture at 30 C for 20 minutes followed by 37 C for 20 minutes (NEB, N0446).

    Gel Extraction:

    Article Title: High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection
    Article Snippet: Linearization of LIC expression vectors for LIC cloning LIC expression vectors (10 µg) were cut with 10 U PmeI in a 20-µL reaction volume and purified from contaminating stuffer fragment and undigested vector by gel-extraction using the NucleoSpin Extract II kit (Macherey & Nagel, Düren, Germany). .. To generate 5′ LIC overhangs (15 and 16 nt, respectively) at both ends the purified vector backbone was treated for 30 min (22°C) with T4 DNA polymerase in the presence of dATP, using the following reaction setup: 0.2 pmol purified vector backbone, 2 µL 10× buffer 2 (NEB), 2 µL dATP (25 mM), 1 µL dithiothreitol (DTT, 100 mM), 2 µL 10× (10 mg/mL) bovine serum albumin (BSA; NEB), 10 U T4 DNA polymerase (NEB) in a volume of 20 µL (filled up with ddH2 O).

    Plasmid Preparation:

    Article Title: High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection
    Article Snippet: .. To generate 5′ LIC overhangs (15 and 16 nt, respectively) at both ends the purified vector backbone was treated for 30 min (22°C) with T4 DNA polymerase in the presence of dATP, using the following reaction setup: 0.2 pmol purified vector backbone, 2 µL 10× buffer 2 (NEB), 2 µL dATP (25 mM), 1 µL dithiothreitol (DTT, 100 mM), 2 µL 10× (10 mg/mL) bovine serum albumin (BSA; NEB), 10 U T4 DNA polymerase (NEB) in a volume of 20 µL (filled up with ddH2 O). .. The reaction mix was heat inactivated for 20 min at 75°C, followed by purification using the NucleoSpin Extract II kit (Macherey & Nagel) and elution with 20 µL elution buffer included in the kit.

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