app dna rna ligase  (New England Biolabs)


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    Name:
    Thermostable 5 App DNA RNA Ligase
    Description:
    Thermostable 5 App DNA RNA Ligase 50 rxns
    Catalog Number:
    m0319l
    Price:
    273
    Size:
    50 rxns
    Category:
    DNA Ligases
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    New England Biolabs app dna rna ligase
    Thermostable 5 App DNA RNA Ligase
    Thermostable 5 App DNA RNA Ligase 50 rxns
    https://www.bioz.com/result/app dna rna ligase/product/New England Biolabs
    Average 90 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    app dna rna ligase - by Bioz Stars, 2020-01
    90/100 stars

    Images

    1) Product Images from "Template-switching mechanism of a group II intron-encoded reverse transcriptase and its implications for biological function and RNA-Seq"

    Article Title: Template-switching mechanism of a group II intron-encoded reverse transcriptase and its implications for biological function and RNA-Seq

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA119.011337

    Models of template switching and non-templated nucleotide addition reactions. A , template switching to an acceptor RNA from an RNA template/DNA primer heteroduplex with a 1-nt 3′ overhang added by NTA after completion of cDNA synthesis or as an artificial starter duplex. B , NTA to a blunt-end RNA/DNA heteroduplex in the absence of an acceptor nucleic acid. C , template-switching to an acceptor RNA from a blunt-end RNA/DNA heteroduplex without NTA. See under “Discussion” for details.
    Figure Legend Snippet: Models of template switching and non-templated nucleotide addition reactions. A , template switching to an acceptor RNA from an RNA template/DNA primer heteroduplex with a 1-nt 3′ overhang added by NTA after completion of cDNA synthesis or as an artificial starter duplex. B , NTA to a blunt-end RNA/DNA heteroduplex in the absence of an acceptor nucleic acid. C , template-switching to an acceptor RNA from a blunt-end RNA/DNA heteroduplex without NTA. See under “Discussion” for details.

    Techniques Used:

    2) Product Images from "Elimination of Ligation Dependent Artifacts in T4 RNA Ligase to Achieve High Efficiency and Low Bias MicroRNA Capture"

    Article Title: Elimination of Ligation Dependent Artifacts in T4 RNA Ligase to Achieve High Efficiency and Low Bias MicroRNA Capture

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0094619

    Comparison of capture efficiency across the 20 microRNA panel spiked into 500 ng of total RNA using 4 different adapter designs. The rA and dA adapters have identical sequences based on modified modban design except the 5′ base is either RNA or DNA as indicated. The SR1 and SR1-S adapters are taken from Zhuang et al (39). T4 Rnl2 TK shows no preference for DNA or RNA at the ligation site. However, overall capture efficiency and bias were significantly worse for the SR1 and SR1-S adapters.
    Figure Legend Snippet: Comparison of capture efficiency across the 20 microRNA panel spiked into 500 ng of total RNA using 4 different adapter designs. The rA and dA adapters have identical sequences based on modified modban design except the 5′ base is either RNA or DNA as indicated. The SR1 and SR1-S adapters are taken from Zhuang et al (39). T4 Rnl2 TK shows no preference for DNA or RNA at the ligation site. However, overall capture efficiency and bias were significantly worse for the SR1 and SR1-S adapters.

    Techniques Used: Modification, Ligation

    MicroRNA capture was performed with 4 different ligases using the vendor recommended protocols to compare capture efficiency across 20 different microRNA. The ligation products were analyzed by 15% denaturing urea-PAGE. Capture efficiency was determined by performing a Cy3 scan and comparing the intensities of the ∼40 nt captured microRNA band versus the ∼20 nt free microRNA band. T4 RNA Ligase 2 truncated (T4 Rnl2 T) had high average capture efficiency and low bias but many randomly sized background products. The point mutant enzymes T4 RNA Ligase 2 truncated K227Q (T4 Rnl2 TK) and T4 RNA Ligase 2 truncated KQ (T4 Rnl2 TKQ) had decreased side product formation but also lower average capture efficiency and higher bias. Thermostable 5′ App DNA/RNA Ligase (Mth Rnl), which was performed at 65°C instead of 25°C, had similar average capture efficiency and bias but with distinct ligation efficiency pattern.
    Figure Legend Snippet: MicroRNA capture was performed with 4 different ligases using the vendor recommended protocols to compare capture efficiency across 20 different microRNA. The ligation products were analyzed by 15% denaturing urea-PAGE. Capture efficiency was determined by performing a Cy3 scan and comparing the intensities of the ∼40 nt captured microRNA band versus the ∼20 nt free microRNA band. T4 RNA Ligase 2 truncated (T4 Rnl2 T) had high average capture efficiency and low bias but many randomly sized background products. The point mutant enzymes T4 RNA Ligase 2 truncated K227Q (T4 Rnl2 TK) and T4 RNA Ligase 2 truncated KQ (T4 Rnl2 TKQ) had decreased side product formation but also lower average capture efficiency and higher bias. Thermostable 5′ App DNA/RNA Ligase (Mth Rnl), which was performed at 65°C instead of 25°C, had similar average capture efficiency and bias but with distinct ligation efficiency pattern.

    Techniques Used: Ligation, Polyacrylamide Gel Electrophoresis, Mutagenesis

    3) Product Images from "Template-switching mechanism of a group II intron-encoded reverse transcriptase and its implications for biological function and RNA-Seq"

    Article Title: Template-switching mechanism of a group II intron-encoded reverse transcriptase and its implications for biological function and RNA-Seq

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA119.011337

    Models of template switching and non-templated nucleotide addition reactions. A , template switching to an acceptor RNA from an RNA template/DNA primer heteroduplex with a 1-nt 3′ overhang added by NTA after completion of cDNA synthesis or as an artificial starter duplex. B , NTA to a blunt-end RNA/DNA heteroduplex in the absence of an acceptor nucleic acid. C , template-switching to an acceptor RNA from a blunt-end RNA/DNA heteroduplex without NTA. See under “Discussion” for details.
    Figure Legend Snippet: Models of template switching and non-templated nucleotide addition reactions. A , template switching to an acceptor RNA from an RNA template/DNA primer heteroduplex with a 1-nt 3′ overhang added by NTA after completion of cDNA synthesis or as an artificial starter duplex. B , NTA to a blunt-end RNA/DNA heteroduplex in the absence of an acceptor nucleic acid. C , template-switching to an acceptor RNA from a blunt-end RNA/DNA heteroduplex without NTA. See under “Discussion” for details.

    Techniques Used:

    Related Articles

    Amplification:

    Article Title: Sequence-specific cleavage of dsRNA by Mini-III RNase
    Article Snippet: The cDNA was purified with a GeneJET DNA Cleanup Micro Kit (Thermo Scientific) and the second preadenylated adaptor PreA3Univ was ligated to the cDNA 3′ ends by Thermostable 5′ App DNA/RNA Ligase (New England Biolabs). .. After removal of unligated adapters with GeneJET DNA Cleanup Micro Kit, the cDNA was amplified in a PCR reaction (15–18 cycles of amplification were used).

    Article Title: BCDIN3D regulates tRNAHis 3’ fragment processing
    Article Snippet: The reactions were then cooled to room temperature and neutralized with 5 N HCl. cDNAs were purified by using a Qiagen MinElute Reaction Cleanup Kit and then ligated at their 3’ ends to a DNA oligonucleotide encoding the reverse complement of the Illumina Read1 primer binding site (R1R) using Thermostable 5’ AppDNA/RNA Ligase (New England Biolabs). .. Ligated cDNAs were re-purified with MinElute Reaction Cleanup Kit and amplified by PCR for 12 cycles using Phusion DNA polymerase (Thermo Fisher Scientific) with overlapping multiplex and barcode primers that add sequences necessary for Illumina sequencing.

    Article Title: Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction
    Article Snippet: The R1R DNA adapter was pre-adenylated by using an adenylation kit (New England Biolabs) and then ligated to the 3′ end of the cDNA by using thermostable 5′ App DNA/RNA Ligase (New England Biolabs) for 2 h at 65 °C. .. The ligated products were purified by using a MinElute Reaction Cleanup Kit and amplified by PCR with Phusion High-Fidelity DNA polymerase (Thermo Fisher Scientific; denaturation at 98 °C for 5 sec followed by 12 cycles of 98 °C 5 sec, 60 °C 10 sec, 72 °C 15 sec and then held at 4 °C).

    Article Title: A highly proliferative group IIC intron from Geobacillus stearothermophilus reveals new features of group II intron mobility and splicing
    Article Snippet: .. After cooling to room temperature, the mixtures were neutralized by adding 1 μl of 5 N HCl, and cDNAs were purified twice with a MinElute Reaction Cleanup Kit (QIAGEN). cDNAs were then ligated to a 5′-adenylated/3′-blocked (C3 spacer, 3SpC3; IDT) adapter (R1R; ) by using thermostable 5′ AppDNA/RNA Ligase (New England Biolabs), and the ligated cDNAs were re-purified with a MinElute Reaction Cleanup Kit (QIAGEN) and amplified with Phusion High-Fidelity DNA polymerase (Thermo Fisher) with 200 nM of Illumina multiplex and 200 nM of barcode primers ( ). .. PCR was done at 98 °C for 5 sec pre-denaturation followed by 12 cycles of 98 °C for 5 sec, 60 °C for 10 sec, and 72 °C for 10 sec.

    Article Title: Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction
    Article Snippet: .. After reverse transcription, a second RNA-seq adapter (R1R DNA; containing the reverse complement of an Illumina Read 1 sequence) is ligated to the opposite end of the cDNA by a single-stranded DNA ligation with thermostable 5′ App RNA/DNA ligase (New England Biolabs), and this is followed by minimal PCR amplification with primers that add Illumina capture sites and sequencing indices. .. By avoiding gel-purification steps, TGIRT-seq libraries can be generated rapidly from small amounts of starting material (1–2 ng input RNA).

    DNA Ligation:

    Article Title: Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction
    Article Snippet: .. After reverse transcription, a second RNA-seq adapter (R1R DNA; containing the reverse complement of an Illumina Read 1 sequence) is ligated to the opposite end of the cDNA by a single-stranded DNA ligation with thermostable 5′ App RNA/DNA ligase (New England Biolabs), and this is followed by minimal PCR amplification with primers that add Illumina capture sites and sequencing indices. .. By avoiding gel-purification steps, TGIRT-seq libraries can be generated rapidly from small amounts of starting material (1–2 ng input RNA).

    Neutralization:

    Article Title: Template-switching mechanism of a group II intron-encoded reverse transcriptase and its implications for biological function and RNA-Seq
    Article Snippet: After terminating the reactions with NaOH and neutralization with HCl as described above for template-switching reactions, the volume was raised to 100 μl with H2 O, and cDNA products containing the R2R adapter attached to their 5′ end were cleaned-up by using a MinElute column (Qiagen) to remove unused primer. .. A 5′-adenylated R1R adapter was then ligated to the 3′ end of the cDNA using Thermostable 5′ App DNA/RNA Ligase (New England Biolabs) for 1 h at 65 °C.

    Article Title: Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction
    Article Snippet: The template-switching reactions were incubated for 15 min at 60 °C and then terminated by adding 1 μl 5 M NaOH to degrade RNA and heating at 95 °C for 5 min followed by neutralization with 1 μl 5 M HCl and two rounds of MinElute column clean-up (Qiagen) to decrease the amount of unused R2R DNA adapter. .. The R1R DNA adapter was pre-adenylated by using an adenylation kit (New England Biolabs) and then ligated to the 3′ end of the cDNA by using thermostable 5′ App DNA/RNA Ligase (New England Biolabs) for 2 h at 65 °C.

    Blocking Assay:

    Article Title: Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction
    Article Snippet: For Illumina RNA-seq, the initial RNA template/DNA primer consists of a 34-nt RNA oligonucleotide containing an Illumina Read 2 sequence (R2 RNA) with a 3′ blocking group (C3 Spacer, 3SpC3) annealed to a 35-nt DNA primer (R2R DNA) that leaves a single nucleotide 3′-DNA overhang. .. After reverse transcription, a second RNA-seq adapter (R1R DNA; containing the reverse complement of an Illumina Read 1 sequence) is ligated to the opposite end of the cDNA by a single-stranded DNA ligation with thermostable 5′ App RNA/DNA ligase (New England Biolabs), and this is followed by minimal PCR amplification with primers that add Illumina capture sites and sequencing indices.

    Incubation:

    Article Title: BCDIN3D regulates tRNAHis 3’ fragment processing
    Article Snippet: Reactions were incubated at 60°C for 15 min and were terminated by adding 5 N NaOH to a final concentration of 0.25 N and incubated at 95°C for 3 min to degrade RNAs and denature protein. .. The reactions were then cooled to room temperature and neutralized with 5 N HCl. cDNAs were purified by using a Qiagen MinElute Reaction Cleanup Kit and then ligated at their 3’ ends to a DNA oligonucleotide encoding the reverse complement of the Illumina Read1 primer binding site (R1R) using Thermostable 5’ AppDNA/RNA Ligase (New England Biolabs).

    Article Title: Archaeal Nucleic Acid Ligases and Their Potential in Biotechnology
    Article Snippet: The adenylated adaptors can then be incubated with the pool of target miRNA molecules and the mutated ligase. .. In order to implement this protocol, the mutated enzyme is commercially available (as the Thermostable 5′ AppDNA/RNA Ligase from New England Biolabs).

    Article Title: Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction
    Article Snippet: The template-switching reactions were incubated for 15 min at 60 °C and then terminated by adding 1 μl 5 M NaOH to degrade RNA and heating at 95 °C for 5 min followed by neutralization with 1 μl 5 M HCl and two rounds of MinElute column clean-up (Qiagen) to decrease the amount of unused R2R DNA adapter. .. The R1R DNA adapter was pre-adenylated by using an adenylation kit (New England Biolabs) and then ligated to the 3′ end of the cDNA by using thermostable 5′ App DNA/RNA Ligase (New England Biolabs) for 2 h at 65 °C.

    Article Title: A highly proliferative group IIC intron from Geobacillus stearothermophilus reveals new features of group II intron mobility and splicing
    Article Snippet: The reverse transcription reaction was stopped by adding 1 μl of 5 N NaOH, and incubated at 95 °C for 3 min to degrade RNA. .. After cooling to room temperature, the mixtures were neutralized by adding 1 μl of 5 N HCl, and cDNAs were purified twice with a MinElute Reaction Cleanup Kit (QIAGEN). cDNAs were then ligated to a 5′-adenylated/3′-blocked (C3 spacer, 3SpC3; IDT) adapter (R1R; ) by using thermostable 5′ AppDNA/RNA Ligase (New England Biolabs), and the ligated cDNAs were re-purified with a MinElute Reaction Cleanup Kit (QIAGEN) and amplified with Phusion High-Fidelity DNA polymerase (Thermo Fisher) with 200 nM of Illumina multiplex and 200 nM of barcode primers ( ).

    Activity Assay:

    Article Title: Elimination of Ligation Dependent Artifacts in T4 RNA Ligase to Achieve High Efficiency and Low Bias MicroRNA Capture
    Article Snippet: Six microRNA were captured at < 2% efficiency.T4 RNA ligase 2 truncated KQ (T4 Rnl 2 TKQ) is a double-point mutant that is also designed to have low side product formation but with increased ligation activity that is restored to the levels of T4 Rnl2 T . .. Finally, we tried Thermostable 5′ App DNA/RNA Ligase (MthRnl) from New England Biolabs, which is a point mutant of RNA ligase isolated from Methanobacterium thermoautotrophicum .

    Modification:

    Article Title: BCDIN3D regulates tRNAHis 3’ fragment processing
    Article Snippet: TGIRT-seq libraries TGIRT-seq libraries were prepared with a modification of the Total RNA-seq method [ ]. .. The reactions were then cooled to room temperature and neutralized with 5 N HCl. cDNAs were purified by using a Qiagen MinElute Reaction Cleanup Kit and then ligated at their 3’ ends to a DNA oligonucleotide encoding the reverse complement of the Illumina Read1 primer binding site (R1R) using Thermostable 5’ AppDNA/RNA Ligase (New England Biolabs).

    Article Title: Template-switching mechanism of a group II intron-encoded reverse transcriptase and its implications for biological function and RNA-Seq
    Article Snippet: These 3′ biases, which are restricted to the 3′-terminal nucleotide of the acceptor, account for about half of the sequence bias in TGIRT-seq, the remainder coming from the Thermostable 5′ App RNA/DNA Ligase (New England Biolabs) used for R1R adapter ligation (see A ) ( ). .. It should also be noted that the 3′-end biases for template switching by GsI–IIC RT (for acceptor RNAs with a 3′-G and against those with a 3′-U) are not universal, as TeI4c RT preferentially template switches to acceptor RNAs with a 3′-A residue, ), as well as more efficient use of RNAs with modified 3′ ends, including 3′-phosphate and 2′- O -Me groups , which are present at the 3′ ends of PIWI-interacting RNAs and plant miRNAs ( , ).

    Gel Purification:

    Article Title: Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction
    Article Snippet: After reverse transcription, a second RNA-seq adapter (R1R DNA; containing the reverse complement of an Illumina Read 1 sequence) is ligated to the opposite end of the cDNA by a single-stranded DNA ligation with thermostable 5′ App RNA/DNA ligase (New England Biolabs), and this is followed by minimal PCR amplification with primers that add Illumina capture sites and sequencing indices. .. By avoiding gel-purification steps, TGIRT-seq libraries can be generated rapidly from small amounts of starting material (1–2 ng input RNA).

    Significance Assay:

    Article Title: A highly proliferative group IIC intron from Geobacillus stearothermophilus reveals new features of group II intron mobility and splicing
    Article Snippet: After cooling to room temperature, the mixtures were neutralized by adding 1 μl of 5 N HCl, and cDNAs were purified twice with a MinElute Reaction Cleanup Kit (QIAGEN). cDNAs were then ligated to a 5′-adenylated/3′-blocked (C3 spacer, 3SpC3; IDT) adapter (R1R; ) by using thermostable 5′ AppDNA/RNA Ligase (New England Biolabs), and the ligated cDNAs were re-purified with a MinElute Reaction Cleanup Kit (QIAGEN) and amplified with Phusion High-Fidelity DNA polymerase (Thermo Fisher) with 200 nM of Illumina multiplex and 200 nM of barcode primers ( ). .. After trimming of Illumina TruSeq adapters and PCR primer sequences with cutadapt [ ] (sequencing quality score cut-off at 20; p-value < 0.01) and discarding reads of ≤7 nt, the reads were mapped to the precursor and expected ligated exons with Bowtie2 v2.2.6 [ ] with local alignment, and custom setting of “ --no-mixed --norc --no-discordant --very-sensitive-local -k 2 --score-min L,-6,1.98 --mp 2” s. Unmapped reads were then remapped to precursor sequences using HISAT2 v2.0.2 [ ] with default settings to capture any reads from alternative splicing.

    Ligation:

    Article Title: Elimination of Ligation Dependent Artifacts in T4 RNA Ligase to Achieve High Efficiency and Low Bias MicroRNA Capture
    Article Snippet: Paragraph title: Ligation Protocol ... In the experiments where different ligases were investigated, T4 RNA Ligase 2 truncated, T4 RNA Ligase 2 truncated R55K K227Q, and Thermostable 5′ App DNA/RNA Ligase were all obtained from New England Biolabs.

    Article Title: Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction
    Article Snippet: .. Here, we used the previously determined ligation biases of the thermostable 5′ App RNA/DNA ligase , , to design an R2/R2R adapter with just a single nucleotide change that dramatically decreases the formation of adapter dimers, thereby improving the recovery of miRNA sequences and enabling the construction of TGIRT-seq libraries from even smaller amounts of starting material. ..

    Article Title: Archaeal Nucleic Acid Ligases and Their Potential in Biotechnology
    Article Snippet: The ability of the M. thermautotrophicus RNA ligase (and the K97A mutant) to function at 65°C also helps to remove ligation bias associated with RNA secondary structures. .. In order to implement this protocol, the mutated enzyme is commercially available (as the Thermostable 5′ AppDNA/RNA Ligase from New England Biolabs).

    Article Title: Detection of spacer precursors formed in vivo during primed CRISPR adaptation
    Article Snippet: .. The two-step ligation procedure described above was repeated using 5 μl of adenylated 3′-ligated DNA fragments, 0.5 μM of barcoded oligos i112, i113, i114 or i115 that served as 5′ adapters (barcodes were used as internal controls; Supplementary Table ), 10 pmol of thermostable 5′ App DNA/RNA ligase at the first ligation step, and 10 U of T4 RNA ligase 1 at the second ligation step. ..

    Article Title: Template-switching mechanism of a group II intron-encoded reverse transcriptase and its implications for biological function and RNA-Seq
    Article Snippet: .. These 3′ biases, which are restricted to the 3′-terminal nucleotide of the acceptor, account for about half of the sequence bias in TGIRT-seq, the remainder coming from the Thermostable 5′ App RNA/DNA Ligase (New England Biolabs) used for R1R adapter ligation (see A ) ( ). ..

    Article Title: Elimination of Ligation Dependent Artifacts in T4 RNA Ligase to Achieve High Efficiency and Low Bias MicroRNA Capture
    Article Snippet: Six microRNA were captured at < 2% efficiency.T4 RNA ligase 2 truncated KQ (T4 Rnl 2 TKQ) is a double-point mutant that is also designed to have low side product formation but with increased ligation activity that is restored to the levels of T4 Rnl2 T . .. Finally, we tried Thermostable 5′ App DNA/RNA Ligase (MthRnl) from New England Biolabs, which is a point mutant of RNA ligase isolated from Methanobacterium thermoautotrophicum .

    Generated:

    Article Title: Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction
    Article Snippet: After reverse transcription, a second RNA-seq adapter (R1R DNA; containing the reverse complement of an Illumina Read 1 sequence) is ligated to the opposite end of the cDNA by a single-stranded DNA ligation with thermostable 5′ App RNA/DNA ligase (New England Biolabs), and this is followed by minimal PCR amplification with primers that add Illumina capture sites and sequencing indices. .. By avoiding gel-purification steps, TGIRT-seq libraries can be generated rapidly from small amounts of starting material (1–2 ng input RNA).

    other:

    Article Title: Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction
    Article Snippet: We found that the 5′-sequence biases introduced mainly by the thermostable 5′ App RNA/DNA ligase could be computationally corrected and that 3′-biases introduced by TGIRT template-switching could be corrected either computationally or by employing an altered ratio of 3′-overhang nucleotides in the R2 RNA/R2R DNA primer mix.

    Sequencing:

    Article Title: BCDIN3D regulates tRNAHis 3’ fragment processing
    Article Snippet: Reverse transcription with TGIRT-III (InGex) was initiated from a DNA primer (5'-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTN-3') encoding the reverse complement of the Illumina Read2 sequencing primer binding site (R2R) annealed to a complementary RNA oligonucleotide (R2) such that there is a single nucleotide 3’ DNA overhang composed of an equimolar mixture of A, G, C and T. The RNA oligonucleotide is blocked at its 3’ end with C3Sp (IDT) to inhibit template switching to itself. .. The reactions were then cooled to room temperature and neutralized with 5 N HCl. cDNAs were purified by using a Qiagen MinElute Reaction Cleanup Kit and then ligated at their 3’ ends to a DNA oligonucleotide encoding the reverse complement of the Illumina Read1 primer binding site (R1R) using Thermostable 5’ AppDNA/RNA Ligase (New England Biolabs).

    Article Title: Detection of spacer precursors formed in vivo during primed CRISPR adaptation
    Article Snippet: Paragraph title: High-throughput sequencing of DNA fragments: FragSeq ... DNA fragments < 700 bp (in 5 μl of water) were heat-denatured at 95 °C for 5 min, cooled to 65 °C, and mixed with 0.5 μM adenylated oligo i116, 1× NEBuffer 1, 5 mM MnCl2 and 10 pmol of thermostable 5′ App DNA/RNA ligase (NEB) in 10-μl reaction volume.

    Article Title: Template-switching mechanism of a group II intron-encoded reverse transcriptase and its implications for biological function and RNA-Seq
    Article Snippet: .. These 3′ biases, which are restricted to the 3′-terminal nucleotide of the acceptor, account for about half of the sequence bias in TGIRT-seq, the remainder coming from the Thermostable 5′ App RNA/DNA Ligase (New England Biolabs) used for R1R adapter ligation (see A ) ( ). ..

    Article Title: A highly proliferative group IIC intron from Geobacillus stearothermophilus reveals new features of group II intron mobility and splicing
    Article Snippet: Paragraph title: High-throughput TGIRT sequencing of splicing products ... After cooling to room temperature, the mixtures were neutralized by adding 1 μl of 5 N HCl, and cDNAs were purified twice with a MinElute Reaction Cleanup Kit (QIAGEN). cDNAs were then ligated to a 5′-adenylated/3′-blocked (C3 spacer, 3SpC3; IDT) adapter (R1R; ) by using thermostable 5′ AppDNA/RNA Ligase (New England Biolabs), and the ligated cDNAs were re-purified with a MinElute Reaction Cleanup Kit (QIAGEN) and amplified with Phusion High-Fidelity DNA polymerase (Thermo Fisher) with 200 nM of Illumina multiplex and 200 nM of barcode primers ( ).

    Article Title: Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction
    Article Snippet: .. After reverse transcription, a second RNA-seq adapter (R1R DNA; containing the reverse complement of an Illumina Read 1 sequence) is ligated to the opposite end of the cDNA by a single-stranded DNA ligation with thermostable 5′ App RNA/DNA ligase (New England Biolabs), and this is followed by minimal PCR amplification with primers that add Illumina capture sites and sequencing indices. .. By avoiding gel-purification steps, TGIRT-seq libraries can be generated rapidly from small amounts of starting material (1–2 ng input RNA).

    Binding Assay:

    Article Title: BCDIN3D regulates tRNAHis 3’ fragment processing
    Article Snippet: .. The reactions were then cooled to room temperature and neutralized with 5 N HCl. cDNAs were purified by using a Qiagen MinElute Reaction Cleanup Kit and then ligated at their 3’ ends to a DNA oligonucleotide encoding the reverse complement of the Illumina Read1 primer binding site (R1R) using Thermostable 5’ AppDNA/RNA Ligase (New England Biolabs). .. Ligated cDNAs were re-purified with MinElute Reaction Cleanup Kit and amplified by PCR for 12 cycles using Phusion DNA polymerase (Thermo Fisher Scientific) with overlapping multiplex and barcode primers that add sequences necessary for Illumina sequencing.

    RNA Sequencing Assay:

    Article Title: Sequence-specific cleavage of dsRNA by Mini-III RNase
    Article Snippet: Paragraph title: RNA sequencing analyses ... The cDNA was purified with a GeneJET DNA Cleanup Micro Kit (Thermo Scientific) and the second preadenylated adaptor PreA3Univ was ligated to the cDNA 3′ ends by Thermostable 5′ App DNA/RNA Ligase (New England Biolabs).

    Article Title: BCDIN3D regulates tRNAHis 3’ fragment processing
    Article Snippet: TGIRT-seq libraries TGIRT-seq libraries were prepared with a modification of the Total RNA-seq method [ ]. .. The reactions were then cooled to room temperature and neutralized with 5 N HCl. cDNAs were purified by using a Qiagen MinElute Reaction Cleanup Kit and then ligated at their 3’ ends to a DNA oligonucleotide encoding the reverse complement of the Illumina Read1 primer binding site (R1R) using Thermostable 5’ AppDNA/RNA Ligase (New England Biolabs).

    Article Title: Template-switching mechanism of a group II intron-encoded reverse transcriptase and its implications for biological function and RNA-Seq
    Article Snippet: Paragraph title: RNA sequencing ... A 5′-adenylated R1R adapter was then ligated to the 3′ end of the cDNA using Thermostable 5′ App DNA/RNA Ligase (New England Biolabs) for 1 h at 65 °C.

    Article Title: Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction
    Article Snippet: .. In this regard, a weakness of the TGIRT Total RNA-seq method is the thermostable 5′ App RNA/DNA ligase used to attach the R1R adapter to the 3′ end of the cDNA, which introduces sampling biases for cDNA ends and produces unwanted adapter dimers by ligating the R1R adapter to residual R2R adapter carried over from previous steps. .. To avoid wasting reads, these adapter dimers are removed by AMPure beads clean-up of the library prior to sequencing (Fig. ), a step that can result in the differential loss of sequences corresponding to miRNAs and other very small RNAs, whose library products are close in size to adapter dimers (146 and 124 nt, respectively) .

    Article Title: Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction
    Article Snippet: .. After reverse transcription, a second RNA-seq adapter (R1R DNA; containing the reverse complement of an Illumina Read 1 sequence) is ligated to the opposite end of the cDNA by a single-stranded DNA ligation with thermostable 5′ App RNA/DNA ligase (New England Biolabs), and this is followed by minimal PCR amplification with primers that add Illumina capture sites and sequencing indices. .. By avoiding gel-purification steps, TGIRT-seq libraries can be generated rapidly from small amounts of starting material (1–2 ng input RNA).

    Mutagenesis:

    Article Title: Archaeal Nucleic Acid Ligases and Their Potential in Biotechnology
    Article Snippet: The ability of the M. thermautotrophicus RNA ligase (and the K97A mutant) to function at 65°C also helps to remove ligation bias associated with RNA secondary structures. .. In order to implement this protocol, the mutated enzyme is commercially available (as the Thermostable 5′ AppDNA/RNA Ligase from New England Biolabs).

    Article Title: Elimination of Ligation Dependent Artifacts in T4 RNA Ligase to Achieve High Efficiency and Low Bias MicroRNA Capture
    Article Snippet: .. Finally, we tried Thermostable 5′ App DNA/RNA Ligase (MthRnl) from New England Biolabs, which is a point mutant of RNA ligase isolated from Methanobacterium thermoautotrophicum . ..

    Isolation:

    Article Title: Elimination of Ligation Dependent Artifacts in T4 RNA Ligase to Achieve High Efficiency and Low Bias MicroRNA Capture
    Article Snippet: .. Finally, we tried Thermostable 5′ App DNA/RNA Ligase (MthRnl) from New England Biolabs, which is a point mutant of RNA ligase isolated from Methanobacterium thermoautotrophicum . ..

    Article Title: A highly proliferative group IIC intron from Geobacillus stearothermophilus reveals new features of group II intron mobility and splicing
    Article Snippet: For analysis of ligated, free, and alternatively spliced exons, splicing reactions were done as described above with 80 nM protein and 40 nM RNA for 15 min at 50 °C, and small RNAs ( < 200 nt) were isolated with an RNA Clean & Concentrator Kit (Zymo Research). .. After cooling to room temperature, the mixtures were neutralized by adding 1 μl of 5 N HCl, and cDNAs were purified twice with a MinElute Reaction Cleanup Kit (QIAGEN). cDNAs were then ligated to a 5′-adenylated/3′-blocked (C3 spacer, 3SpC3; IDT) adapter (R1R; ) by using thermostable 5′ AppDNA/RNA Ligase (New England Biolabs), and the ligated cDNAs were re-purified with a MinElute Reaction Cleanup Kit (QIAGEN) and amplified with Phusion High-Fidelity DNA polymerase (Thermo Fisher) with 200 nM of Illumina multiplex and 200 nM of barcode primers ( ).

    Size-exclusion Chromatography:

    Article Title: Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction
    Article Snippet: The R1R DNA adapter was pre-adenylated by using an adenylation kit (New England Biolabs) and then ligated to the 3′ end of the cDNA by using thermostable 5′ App DNA/RNA Ligase (New England Biolabs) for 2 h at 65 °C. .. The ligated products were purified by using a MinElute Reaction Cleanup Kit and amplified by PCR with Phusion High-Fidelity DNA polymerase (Thermo Fisher Scientific; denaturation at 98 °C for 5 sec followed by 12 cycles of 98 °C 5 sec, 60 °C 10 sec, 72 °C 15 sec and then held at 4 °C).

    Article Title: A highly proliferative group IIC intron from Geobacillus stearothermophilus reveals new features of group II intron mobility and splicing
    Article Snippet: After cooling to room temperature, the mixtures were neutralized by adding 1 μl of 5 N HCl, and cDNAs were purified twice with a MinElute Reaction Cleanup Kit (QIAGEN). cDNAs were then ligated to a 5′-adenylated/3′-blocked (C3 spacer, 3SpC3; IDT) adapter (R1R; ) by using thermostable 5′ AppDNA/RNA Ligase (New England Biolabs), and the ligated cDNAs were re-purified with a MinElute Reaction Cleanup Kit (QIAGEN) and amplified with Phusion High-Fidelity DNA polymerase (Thermo Fisher) with 200 nM of Illumina multiplex and 200 nM of barcode primers ( ). .. PCR was done at 98 °C for 5 sec pre-denaturation followed by 12 cycles of 98 °C for 5 sec, 60 °C for 10 sec, and 72 °C for 10 sec.

    Purification:

    Article Title: Sequence-specific cleavage of dsRNA by Mini-III RNase
    Article Snippet: .. The cDNA was purified with a GeneJET DNA Cleanup Micro Kit (Thermo Scientific) and the second preadenylated adaptor PreA3Univ was ligated to the cDNA 3′ ends by Thermostable 5′ App DNA/RNA Ligase (New England Biolabs). .. After removal of unligated adapters with GeneJET DNA Cleanup Micro Kit, the cDNA was amplified in a PCR reaction (15–18 cycles of amplification were used).

    Article Title: BCDIN3D regulates tRNAHis 3’ fragment processing
    Article Snippet: .. The reactions were then cooled to room temperature and neutralized with 5 N HCl. cDNAs were purified by using a Qiagen MinElute Reaction Cleanup Kit and then ligated at their 3’ ends to a DNA oligonucleotide encoding the reverse complement of the Illumina Read1 primer binding site (R1R) using Thermostable 5’ AppDNA/RNA Ligase (New England Biolabs). .. Ligated cDNAs were re-purified with MinElute Reaction Cleanup Kit and amplified by PCR for 12 cycles using Phusion DNA polymerase (Thermo Fisher Scientific) with overlapping multiplex and barcode primers that add sequences necessary for Illumina sequencing.

    Article Title: Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction
    Article Snippet: The R1R DNA adapter was pre-adenylated by using an adenylation kit (New England Biolabs) and then ligated to the 3′ end of the cDNA by using thermostable 5′ App DNA/RNA Ligase (New England Biolabs) for 2 h at 65 °C. .. The ligated products were purified by using a MinElute Reaction Cleanup Kit and amplified by PCR with Phusion High-Fidelity DNA polymerase (Thermo Fisher Scientific; denaturation at 98 °C for 5 sec followed by 12 cycles of 98 °C 5 sec, 60 °C 10 sec, 72 °C 15 sec and then held at 4 °C).

    Article Title: Detection of spacer precursors formed in vivo during primed CRISPR adaptation
    Article Snippet: High-throughput sequencing of DNA fragments: FragSeq The DNA oligo i116 that served as a 3′ adapter was adenylated using 5′ DNA Adenylation Kit (NEB), purified by ethanol precipitation as above and diluted to 10 μM with nuclease-free water (Supplementary Table ). .. DNA fragments < 700 bp (in 5 μl of water) were heat-denatured at 95 °C for 5 min, cooled to 65 °C, and mixed with 0.5 μM adenylated oligo i116, 1× NEBuffer 1, 5 mM MnCl2 and 10 pmol of thermostable 5′ App DNA/RNA ligase (NEB) in 10-μl reaction volume.

    Article Title: A highly proliferative group IIC intron from Geobacillus stearothermophilus reveals new features of group II intron mobility and splicing
    Article Snippet: .. After cooling to room temperature, the mixtures were neutralized by adding 1 μl of 5 N HCl, and cDNAs were purified twice with a MinElute Reaction Cleanup Kit (QIAGEN). cDNAs were then ligated to a 5′-adenylated/3′-blocked (C3 spacer, 3SpC3; IDT) adapter (R1R; ) by using thermostable 5′ AppDNA/RNA Ligase (New England Biolabs), and the ligated cDNAs were re-purified with a MinElute Reaction Cleanup Kit (QIAGEN) and amplified with Phusion High-Fidelity DNA polymerase (Thermo Fisher) with 200 nM of Illumina multiplex and 200 nM of barcode primers ( ). .. PCR was done at 98 °C for 5 sec pre-denaturation followed by 12 cycles of 98 °C for 5 sec, 60 °C for 10 sec, and 72 °C for 10 sec.

    Polymerase Chain Reaction:

    Article Title: Sequence-specific cleavage of dsRNA by Mini-III RNase
    Article Snippet: The cDNA was purified with a GeneJET DNA Cleanup Micro Kit (Thermo Scientific) and the second preadenylated adaptor PreA3Univ was ligated to the cDNA 3′ ends by Thermostable 5′ App DNA/RNA Ligase (New England Biolabs). .. After removal of unligated adapters with GeneJET DNA Cleanup Micro Kit, the cDNA was amplified in a PCR reaction (15–18 cycles of amplification were used).

    Article Title: BCDIN3D regulates tRNAHis 3’ fragment processing
    Article Snippet: The reactions were then cooled to room temperature and neutralized with 5 N HCl. cDNAs were purified by using a Qiagen MinElute Reaction Cleanup Kit and then ligated at their 3’ ends to a DNA oligonucleotide encoding the reverse complement of the Illumina Read1 primer binding site (R1R) using Thermostable 5’ AppDNA/RNA Ligase (New England Biolabs). .. Ligated cDNAs were re-purified with MinElute Reaction Cleanup Kit and amplified by PCR for 12 cycles using Phusion DNA polymerase (Thermo Fisher Scientific) with overlapping multiplex and barcode primers that add sequences necessary for Illumina sequencing.

    Article Title: Template-switching mechanism of a group II intron-encoded reverse transcriptase and its implications for biological function and RNA-Seq
    Article Snippet: A 5′-adenylated R1R adapter was then ligated to the 3′ end of the cDNA using Thermostable 5′ App DNA/RNA Ligase (New England Biolabs) for 1 h at 65 °C. .. After another MinElute clean up, the entirety of the eluent was used for a 12-cycle PCR using Phusion polymerase (Thermo Fisher Scientific), and the resulting libraries were cleaned up by using 1.4× Ampure XP beads to remove residual primers, primer dimers, salts, and enzymes.

    Article Title: Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction
    Article Snippet: The R1R DNA adapter was pre-adenylated by using an adenylation kit (New England Biolabs) and then ligated to the 3′ end of the cDNA by using thermostable 5′ App DNA/RNA Ligase (New England Biolabs) for 2 h at 65 °C. .. The ligated products were purified by using a MinElute Reaction Cleanup Kit and amplified by PCR with Phusion High-Fidelity DNA polymerase (Thermo Fisher Scientific; denaturation at 98 °C for 5 sec followed by 12 cycles of 98 °C 5 sec, 60 °C 10 sec, 72 °C 15 sec and then held at 4 °C).

    Article Title: A highly proliferative group IIC intron from Geobacillus stearothermophilus reveals new features of group II intron mobility and splicing
    Article Snippet: After cooling to room temperature, the mixtures were neutralized by adding 1 μl of 5 N HCl, and cDNAs were purified twice with a MinElute Reaction Cleanup Kit (QIAGEN). cDNAs were then ligated to a 5′-adenylated/3′-blocked (C3 spacer, 3SpC3; IDT) adapter (R1R; ) by using thermostable 5′ AppDNA/RNA Ligase (New England Biolabs), and the ligated cDNAs were re-purified with a MinElute Reaction Cleanup Kit (QIAGEN) and amplified with Phusion High-Fidelity DNA polymerase (Thermo Fisher) with 200 nM of Illumina multiplex and 200 nM of barcode primers ( ). .. PCR was done at 98 °C for 5 sec pre-denaturation followed by 12 cycles of 98 °C for 5 sec, 60 °C for 10 sec, and 72 °C for 10 sec.

    Article Title: Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction
    Article Snippet: .. After reverse transcription, a second RNA-seq adapter (R1R DNA; containing the reverse complement of an Illumina Read 1 sequence) is ligated to the opposite end of the cDNA by a single-stranded DNA ligation with thermostable 5′ App RNA/DNA ligase (New England Biolabs), and this is followed by minimal PCR amplification with primers that add Illumina capture sites and sequencing indices. .. By avoiding gel-purification steps, TGIRT-seq libraries can be generated rapidly from small amounts of starting material (1–2 ng input RNA).

    Gel Extraction:

    Article Title: Sequence-specific cleavage of dsRNA by Mini-III RNase
    Article Snippet: The cDNA was purified with a GeneJET DNA Cleanup Micro Kit (Thermo Scientific) and the second preadenylated adaptor PreA3Univ was ligated to the cDNA 3′ ends by Thermostable 5′ App DNA/RNA Ligase (New England Biolabs). .. The PCR products were resolved in the agarose gel electrophoresis where fraction of size between 200 and 700 bp was selected, purified from agarose gel slices with GeneJET Gel Extraction Kit (Thermo Scientific) and subjected to Next-Generation Sequencing on MiSeq (Illumina) platform at Genomed.

    Chromatin Immunoprecipitation:

    Article Title: Template-switching mechanism of a group II intron-encoded reverse transcriptase and its implications for biological function and RNA-Seq
    Article Snippet: A 5′-adenylated R1R adapter was then ligated to the 3′ end of the cDNA using Thermostable 5′ App DNA/RNA Ligase (New England Biolabs) for 1 h at 65 °C. .. The quality of the libraries was assessed by using a 2100 Bioanalzyer Instrument with a High Sensitivity DNA chip (Agilent).

    Article Title: A highly proliferative group IIC intron from Geobacillus stearothermophilus reveals new features of group II intron mobility and splicing
    Article Snippet: After assessment and quantification on a 2100 Bioanalyzer (Agilent) using a Small RNA chip, small RNAs were analyzed by TGIRT-seq, as described [ ]. .. After cooling to room temperature, the mixtures were neutralized by adding 1 μl of 5 N HCl, and cDNAs were purified twice with a MinElute Reaction Cleanup Kit (QIAGEN). cDNAs were then ligated to a 5′-adenylated/3′-blocked (C3 spacer, 3SpC3; IDT) adapter (R1R; ) by using thermostable 5′ AppDNA/RNA Ligase (New England Biolabs), and the ligated cDNAs were re-purified with a MinElute Reaction Cleanup Kit (QIAGEN) and amplified with Phusion High-Fidelity DNA polymerase (Thermo Fisher) with 200 nM of Illumina multiplex and 200 nM of barcode primers ( ).

    Multiplex Assay:

    Article Title: BCDIN3D regulates tRNAHis 3’ fragment processing
    Article Snippet: The reactions were then cooled to room temperature and neutralized with 5 N HCl. cDNAs were purified by using a Qiagen MinElute Reaction Cleanup Kit and then ligated at their 3’ ends to a DNA oligonucleotide encoding the reverse complement of the Illumina Read1 primer binding site (R1R) using Thermostable 5’ AppDNA/RNA Ligase (New England Biolabs). .. Ligated cDNAs were re-purified with MinElute Reaction Cleanup Kit and amplified by PCR for 12 cycles using Phusion DNA polymerase (Thermo Fisher Scientific) with overlapping multiplex and barcode primers that add sequences necessary for Illumina sequencing.

    Article Title: A highly proliferative group IIC intron from Geobacillus stearothermophilus reveals new features of group II intron mobility and splicing
    Article Snippet: .. After cooling to room temperature, the mixtures were neutralized by adding 1 μl of 5 N HCl, and cDNAs were purified twice with a MinElute Reaction Cleanup Kit (QIAGEN). cDNAs were then ligated to a 5′-adenylated/3′-blocked (C3 spacer, 3SpC3; IDT) adapter (R1R; ) by using thermostable 5′ AppDNA/RNA Ligase (New England Biolabs), and the ligated cDNAs were re-purified with a MinElute Reaction Cleanup Kit (QIAGEN) and amplified with Phusion High-Fidelity DNA polymerase (Thermo Fisher) with 200 nM of Illumina multiplex and 200 nM of barcode primers ( ). .. PCR was done at 98 °C for 5 sec pre-denaturation followed by 12 cycles of 98 °C for 5 sec, 60 °C for 10 sec, and 72 °C for 10 sec.

    Agarose Gel Electrophoresis:

    Article Title: Sequence-specific cleavage of dsRNA by Mini-III RNase
    Article Snippet: The cDNA was purified with a GeneJET DNA Cleanup Micro Kit (Thermo Scientific) and the second preadenylated adaptor PreA3Univ was ligated to the cDNA 3′ ends by Thermostable 5′ App DNA/RNA Ligase (New England Biolabs). .. The PCR products were resolved in the agarose gel electrophoresis where fraction of size between 200 and 700 bp was selected, purified from agarose gel slices with GeneJET Gel Extraction Kit (Thermo Scientific) and subjected to Next-Generation Sequencing on MiSeq (Illumina) platform at Genomed.

    Ethanol Precipitation:

    Article Title: Detection of spacer precursors formed in vivo during primed CRISPR adaptation
    Article Snippet: High-throughput sequencing of DNA fragments: FragSeq The DNA oligo i116 that served as a 3′ adapter was adenylated using 5′ DNA Adenylation Kit (NEB), purified by ethanol precipitation as above and diluted to 10 μM with nuclease-free water (Supplementary Table ). .. DNA fragments < 700 bp (in 5 μl of water) were heat-denatured at 95 °C for 5 min, cooled to 65 °C, and mixed with 0.5 μM adenylated oligo i116, 1× NEBuffer 1, 5 mM MnCl2 and 10 pmol of thermostable 5′ App DNA/RNA ligase (NEB) in 10-μl reaction volume.

    Next-Generation Sequencing:

    Article Title: Sequence-specific cleavage of dsRNA by Mini-III RNase
    Article Snippet: The cDNA was purified with a GeneJET DNA Cleanup Micro Kit (Thermo Scientific) and the second preadenylated adaptor PreA3Univ was ligated to the cDNA 3′ ends by Thermostable 5′ App DNA/RNA Ligase (New England Biolabs). .. The PCR products were resolved in the agarose gel electrophoresis where fraction of size between 200 and 700 bp was selected, purified from agarose gel slices with GeneJET Gel Extraction Kit (Thermo Scientific) and subjected to Next-Generation Sequencing on MiSeq (Illumina) platform at Genomed.

    Article Title: BCDIN3D regulates tRNAHis 3’ fragment processing
    Article Snippet: The reactions were then cooled to room temperature and neutralized with 5 N HCl. cDNAs were purified by using a Qiagen MinElute Reaction Cleanup Kit and then ligated at their 3’ ends to a DNA oligonucleotide encoding the reverse complement of the Illumina Read1 primer binding site (R1R) using Thermostable 5’ AppDNA/RNA Ligase (New England Biolabs). .. Libraries were sequenced on a NextSeq 500 instrument (75-nt, paired-end reads) at the Next Generation Sequencing Facility at MD Anderson Science Park.

    Sampling:

    Article Title: Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction
    Article Snippet: .. In this regard, a weakness of the TGIRT Total RNA-seq method is the thermostable 5′ App RNA/DNA ligase used to attach the R1R adapter to the 3′ end of the cDNA, which introduces sampling biases for cDNA ends and produces unwanted adapter dimers by ligating the R1R adapter to residual R2R adapter carried over from previous steps. .. To avoid wasting reads, these adapter dimers are removed by AMPure beads clean-up of the library prior to sequencing (Fig. ), a step that can result in the differential loss of sequences corresponding to miRNAs and other very small RNAs, whose library products are close in size to adapter dimers (146 and 124 nt, respectively) .

    Article Title: Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction
    Article Snippet: After reverse transcription, a second RNA-seq adapter (R1R DNA; containing the reverse complement of an Illumina Read 1 sequence) is ligated to the opposite end of the cDNA by a single-stranded DNA ligation with thermostable 5′ App RNA/DNA ligase (New England Biolabs), and this is followed by minimal PCR amplification with primers that add Illumina capture sites and sequencing indices. .. In this regard, a weakness of the TGIRT Total RNA-seq method is the thermostable 5′ App RNA/DNA ligase used to attach the R1R adapter to the 3′ end of the cDNA, which introduces sampling biases for cDNA ends and produces unwanted adapter dimers by ligating the R1R adapter to residual R2R adapter carried over from previous steps.

    Concentration Assay:

    Article Title: Sequence-specific cleavage of dsRNA by Mini-III RNase
    Article Snippet: The unligated adapters were removed on GeneJET RNA Cleanup and Concentration Micro columns (Thermo Scientific) and purified RNA was used as a template for the reverse transcription reaction with Maxima reverse transcriptase (Thermo Scientific) and the primer UniShRT complementary to the adapter UniShPreA. .. The cDNA was purified with a GeneJET DNA Cleanup Micro Kit (Thermo Scientific) and the second preadenylated adaptor PreA3Univ was ligated to the cDNA 3′ ends by Thermostable 5′ App DNA/RNA Ligase (New England Biolabs).

    Article Title: BCDIN3D regulates tRNAHis 3’ fragment processing
    Article Snippet: Reactions were incubated at 60°C for 15 min and were terminated by adding 5 N NaOH to a final concentration of 0.25 N and incubated at 95°C for 3 min to degrade RNAs and denature protein. .. The reactions were then cooled to room temperature and neutralized with 5 N HCl. cDNAs were purified by using a Qiagen MinElute Reaction Cleanup Kit and then ligated at their 3’ ends to a DNA oligonucleotide encoding the reverse complement of the Illumina Read1 primer binding site (R1R) using Thermostable 5’ AppDNA/RNA Ligase (New England Biolabs).

    Article Title: A highly proliferative group IIC intron from Geobacillus stearothermophilus reveals new features of group II intron mobility and splicing
    Article Snippet: After pre-incubating at room temperature for 30 min, reactions were initiated by adding 0.8 μl 25 mM dNTPs (final concentration 1 mM dATP, dCTP, dGTP, and dTTP) and incubated for 15 min at 60 °C. .. After cooling to room temperature, the mixtures were neutralized by adding 1 μl of 5 N HCl, and cDNAs were purified twice with a MinElute Reaction Cleanup Kit (QIAGEN). cDNAs were then ligated to a 5′-adenylated/3′-blocked (C3 spacer, 3SpC3; IDT) adapter (R1R; ) by using thermostable 5′ AppDNA/RNA Ligase (New England Biolabs), and the ligated cDNAs were re-purified with a MinElute Reaction Cleanup Kit (QIAGEN) and amplified with Phusion High-Fidelity DNA polymerase (Thermo Fisher) with 200 nM of Illumina multiplex and 200 nM of barcode primers ( ).

    High Throughput Screening Assay:

    Article Title: Detection of spacer precursors formed in vivo during primed CRISPR adaptation
    Article Snippet: Paragraph title: High-throughput sequencing of DNA fragments: FragSeq ... DNA fragments < 700 bp (in 5 μl of water) were heat-denatured at 95 °C for 5 min, cooled to 65 °C, and mixed with 0.5 μM adenylated oligo i116, 1× NEBuffer 1, 5 mM MnCl2 and 10 pmol of thermostable 5′ App DNA/RNA ligase (NEB) in 10-μl reaction volume.

    Article Title: A highly proliferative group IIC intron from Geobacillus stearothermophilus reveals new features of group II intron mobility and splicing
    Article Snippet: Paragraph title: High-throughput TGIRT sequencing of splicing products ... After cooling to room temperature, the mixtures were neutralized by adding 1 μl of 5 N HCl, and cDNAs were purified twice with a MinElute Reaction Cleanup Kit (QIAGEN). cDNAs were then ligated to a 5′-adenylated/3′-blocked (C3 spacer, 3SpC3; IDT) adapter (R1R; ) by using thermostable 5′ AppDNA/RNA Ligase (New England Biolabs), and the ligated cDNAs were re-purified with a MinElute Reaction Cleanup Kit (QIAGEN) and amplified with Phusion High-Fidelity DNA polymerase (Thermo Fisher) with 200 nM of Illumina multiplex and 200 nM of barcode primers ( ).

    Staining:

    Article Title: Detection of spacer precursors formed in vivo during primed CRISPR adaptation
    Article Snippet: DNA fragments < 700 bp (in 5 μl of water) were heat-denatured at 95 °C for 5 min, cooled to 65 °C, and mixed with 0.5 μM adenylated oligo i116, 1× NEBuffer 1, 5 mM MnCl2 and 10 pmol of thermostable 5′ App DNA/RNA ligase (NEB) in 10-μl reaction volume. .. The gel was stained with SYBR Gold nucleic acid gel stain, bands were visualized on a UV transilluminator, and products of ~40 to ~500 nt were excised from the gel and recovered as described in Vvedenskaya et al. .

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