T4 rna ligase 1, by New England Biolabs - Product details

1

cDNA Adapter Ligation for PCR Amplification

Check if the same lab product or an alternative is used in the 5 most similar protocols

Example 7

With reference to FIG. 15, to facilitate PCR amplification, a single-stranded DNA adapter oligo (BC_0047) can be ligated to the 3′ end of cDNA. To prevent concatemers of the adapter oligo, dideoxycytidine (ddC) can be included at the 3′ end of the adapter oligo. BC_0047 was generated with a phosphate at the 5′ end and ddC at the 3′ end. Several enzymes are capable of ligating single-stranded oligo to the 3′ end of single-stranded DNA. Herein, T 4 RNA ligase 1 (NEW ENGLAND BIOLABS®) was used. Thermostable 5′ AppDNA/RNA Ligase (NEW ENGLAND BIOLABS®) can also be used with a preadenylated adaptor oligo.

Specifically, 20 μl of the RNase-treated beads can be added to a single PCR tube. 80 μl of ligase mix (5 μl T 4 RNA ligase 1 (NEW ENGLAND BIOLABS®), 10 μl 10× T4 RNA ligase buffer, 5 μl BC_0047 oligo at 50 μM, 50 μl 50% PEG 8000, and 10 μl 10 mM ATP) can be added to the 20 μl of beads in the PCR tube. 50 μl of the ligase mixed with the beads can be transferred into a new PCR tube to prevent too many beads from settling to the bottom of a single tube and the sample can be incubated at 25° C. for 16 hours.

US11168355B2. Methods and kits for labeling cellular molecules (2021-11-09). University of Washington [US]. Inventors: Georg Seelig [US], Richard Muscat [GB], Alexander B. Rosenberg [US].

+ Open protocol

+ Expand

2

Mature miRNA Sequencing Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols

Ten micrograms of total RNA were combined with 50ng of either the RNA Linker Oligo or the 5’P-RNA Linker Oligo (Supplemental Table 1) for ligation and RT-PCR amplification of mature miRNA molecules as previously described, with minor modifications (Diebel et al., 2010 (link)). Briefly, the RNA-Oligo mixtures were combined with 50U of RNasin (New England BioLabs), 10U of T4 RNA Ligase 1 (New England Biolabs), 5μl of 10× T4 RNA Ligase 1 reaction buffer and brought up to 50μl total volume with nuclease-free water. The ligated RNA was then ethanol precipitated and resuspended in 100μl of nuclease-free water. 200ng of the ligated RNA was used as template for RT-PCR amplification with the following cycling conditions: 30 minutes at 50°C, 15 minutes at 95°C, 35 amplification cycles of 30 seconds at 94°C followed by 30 seconds at 48°C followed by 15 seconds at 72°C, and a single final incubation for 2 minutes and 30 seconds at 72°C. 18μl of the RT-PCR product was run on a 3% TAE-agarose gel and RT-PCR products were visualized by ethidium bromide staining. Primer sequences used in the RT-PCR amplifications are listed in Supplemental Table 1. Sequence confirmation of maturely processed miRNA products was conducted by cloning 4μl of the RT-PCR reaction using the TOPO TA Cloning Kit for Sequencing (Invitrogen).

Diebel K.W., Claypool D.J, & van Dyk L.F. (2014). A conserved RNA polymerase III promoter required for gammaherpesvirus TMER transcription and microRNA processing. Gene, 544(1), 8-18.

+ Open protocol

+ Expand

3

Determining mRNA Terminal Sequences of traA Gene

Check if the same lab product or an alternative is used in the 5 most similar protocols

To determine the 5′-terminal sequence of traA mRNA, cDNA was synthesized using total RNA of Anc(C) as the template, SuperScript®; III reverse transcriptase (Life Technologies, Carlsbad, CA, USA), and traA_r primer. The 5′-phosphorylated DNA linker 5PpACYC_rev was ligated at the 3′-terminus of the first strand cDNA with T4 RNA ligase 1 (New England Biolabs). PCR was performed using the resultant cDNA as the template, PrimeSTAR®; HS DNA polymerase (Takara Bio Inc., Shiga, Japan), and the primers pACYC_rev2 and traA_r2. To determine the 3′-terminal sequence of traA mRNA, universal miRNA cloning linker (5′-rAppCTGTAGGCACCATCAAT-NH2-3′; New England Biolabs) was ligated with the 3′-terminus of total RNA of Anc(C) using T4 RNA ligase 1 (New England Biolabs). The first strand cDNA was synthesized using the primer Linker_r and SuperScript®; III reverse transcriptase (Life Technologies), and then purified cDNA was subjected to PCR using the primers Linker_r and traA_f. Three and two bands of PCR products for 5′- and 3′-terminal sequence determination, respectively, were sliced from the gel and subcloned using a Zero Blunt®; TOPO®; PCR Cloning Kit for Sequencing (Life Technologies). Three to six clones were randomly picked and sequenced by the dideoxynucleotide chain termination sequencing method.

Kashiwagi A., Kitamura H, & Sano Tsushima F. (2015). Characterization of a single mutation in TraQ in a strain of Escherichia coli partially resistant to Qβ infection. Frontiers in Microbiology, 6, 124.

+ Open protocol

+ Expand

4

Single-Stranded DNA Adapter Ligation for PCR

Check if the same lab product or an alternative is used in the 5 most similar protocols

Example 7

With reference to FIG. 15, to facilitate PCR amplification, a single-stranded DNA adapter oligo (BC_0047) can be ligated to the 3′ end of cDNA. To prevent concatemers of the adapter oligo, dideoxycytidine (ddC) can be included at the 3′ end of the adapter oligo. BC_0047 was generated with a phosphate at the 5′ end and ddC at the 3′ end. Several enzymes are capable of ligating single-stranded oligo to the 3′ end of single-stranded DNA. Herein, T 4 RNA ligase 1 (NEW ENGLAND BIOLABS®) was used. Thermostable 5′ AppDNA/RNA Ligase (NEW ENGLAND BIOLABS®) can also be used with a preadenylated adaptor oligo.

Specifically, 20 μl of the RNase-treated beads can be added to a single PCR tube. 80 μl of ligase mix (5 μl T 4 RNA ligase 1 (NEW ENGLAND BIOLABS®), 10 μl 10× T4 RNA ligase buffer, 5 μl BC_0047 oligo at 50 μM, 50 μl 50% PEG 8000, and 10 μl 10 mM ATP) can be added to the 20 μl of beads in the PCR tube. 50 μl of the ligase mixed with the beads can be transferred into a new PCR tube to prevent too many beads from settling to the bottom of a single tube and the sample can be incubated at 25° C. for 16 hours.

US11555216B2. Methods and kits for labeling cellular molecules (2023-01-17). University of Washington [US]. Inventors: Georg Seelig [US], Richard Muscat [GB], Alexander B. Rosenberg [US].

+ Open protocol

+ Expand

5

Single-Stranded DNA Adapter Ligation for PCR

Check if the same lab product or an alternative is used in the 5 most similar protocols

Example 7

With reference to FIG. 15, to facilitate PCR amplification, a single-stranded DNA adapter oligo (BC_0047) can be ligated to the 3′ end of cDNA. To prevent concatemers of the adapter oligo, dideoxycytidine (ddC) can be included at the 3′ end of the adapter oligo. BC_0047 was generated with a phosphate at the 5′ end and ddC at the 3′ end. Several enzymes are capable of ligating single-stranded oligo to the 3′ end of single-stranded DNA. Herein, T 4 RNA ligase 1 (NEW ENGLAND BIOLABS®) was used. Thermostable 5′ AppDNA/RNA Ligase (NEW ENGLAND BIOLABS®) can also be used with a preadenylated adaptor oligo.

Specifically, 20 μl of the RNase-treated beads can be added to a single PCR tube. 80 μl of ligase mix (5 μl T 4 RNA ligase 1 (NEW ENGLAND BIOLABS®), 10 μl 10× T4 RNA ligase buffer, 5 μl BC_0047 oligo at 50 μM, 50 μl 50% PEG 8000, and 10 μl 10 mM ATP) can be added to the 20 μl of beads in the PCR tube. 50 μl of the ligase mixed with the beads can be transferred into a new PCR tube to prevent too many beads from settling to the bottom of a single tube and the sample can be incubated at 25° C. for 16 hours.

US11634751B2. Methods and kits for labeling cellular molecules (2023-04-25). University of Washington [US]. Inventors: Georg Seelig [US], Richard Muscat [GB], Alexander B. Rosenberg [US].

+ Open protocol

+ Expand

6

Single-Stranded DNA Adapter Ligation for PCR

Check if the same lab product or an alternative is used in the 5 most similar protocols

Example 7

With reference to FIG. 15, to facilitate PCR amplification, a single-stranded DNA adapter oligo (BC_0047) can be ligated to the 3′ end of cDNA. To prevent concatemers of the adapter oligo, dideoxycytidine (ddC) can be included at the 3′ end of the adapter oligo. BC_0047 was generated with a phosphate at the 5′ end and ddC at the 3′ end. Several enzymes are capable of ligating single-stranded oligo to the 3′ end of single-stranded DNA. Herein, T4 RNA ligase 1 (NEW ENGLAND BIOLABS®) was used. Thermostable 5′ AppDNA/RNA ligase (NEW ENGLAND BIOLABS®) can also be used with a preadenylated adaptor oligo.

Specifically, 20 μl of the RNase-treated beads can be added to a single PCR tube. 80 μl of ligase mix (5 μl T4 RNA ligase 1 (NEW ENGLAND BIOLABS®), 10 μl 10×T4 RNA ligase buffer, 5 μl BC_0047 oligo at 50 μM, 50 μl 50% PEG 8000, and 10 μl 10 mM ATP) can be added to the 20 μl of beads in the PCR tube. 50 μl of the ligase mixed with the beads can be transferred into a new PCR tube to prevent too many beads from settling to the bottom of a single tube and the sample can be incubated at 25° C. for 16 hours.

US11680283B2. In situ combinatorial labeling of cellular molecules (2023-06-20). UNIVERSITY OF WASHINGTON [US]. Inventors: Georg Seelig [US], Alexander B. Rosenberg [US], Charles Roco [US].

+ Open protocol

+ Expand

7

Single-Stranded DNA Adapter Ligation

Check if the same lab product or an alternative is used in the 5 most similar protocols

Example 7

With reference to FIG. 15, to facilitate PCR amplification, a single-stranded DNA adapter oligo (BC_0047) can be ligated to the 3′ end of cDNA. To prevent concatemers of the adapter oligo, dideoxycytidine (ddC) can be included at the 3′ end of the adapter oligo. BC_0047 was generated with a phosphate at the 5′ end and ddC at the 3′ end. Several enzymes are capable of ligating single-stranded oligo to the 3′ end of single-stranded DNA. Herein, T 4 RNA ligase 1 (NEW ENGLAND BIOLABS®) was used. Thermostable 5′ AppDNA/RNA Ligase (NEW ENGLAND BIOLABS®) can also be used with a preadenylated adaptor oligo.

Specifically, 20 μl of the RNase-treated beads can be added to a single PCR tube. 80 μl of ligase mix (5 μl T 4 RNA ligase 1 (NEW ENGLAND BIOLABS®), 10 μl 10× T4 RNA ligase buffer, 5 μl BC_0047 oligo at 50 μM, 50 μl 50% PEG 8000, and 10 μl 10 mM ATP) can be added to the 20 μl of beads in the PCR tube. 50 μl of the ligase mixed with the beads can be transferred into a new PCR tube to prevent too many beads from settling to the bottom of a single tube and the sample can be incubated at 25° C. for 16 hours.

US11639519B1. Methods and kits for labeling cellular molecules (2023-05-02). University of Washington [US]. Inventors: Georg Seelig [US], Richard Muscat [GB], Alexander B. Rosenberg [US].

+ Open protocol

+ Expand

8

3' cDNA Adapter Ligation

Check if the same lab product or an alternative is used in the 5 most similar protocols

Example 7

With reference to FIG. 15, to facilitate PCR amplification, a single-stranded DNA adapter oligo (BC_0047) can be ligated to the 3′ end of cDNA. To prevent concatemers of the adapter oligo, dideoxycytidine (ddC) can be included at the 3′ end of the adapter oligo. BC_0047 was generated with a phosphate at the 5′ end and ddC at the 3′ end. Several enzymes are capable of ligating single-stranded oligo to the 3′ end of single-stranded DNA. Herein, T4 RNA ligase 1 (NEW ENGLAND BIOLABS®) was used. Thermostable 5′ AppDNA/RNA ligase (NEW ENGLAND BIOLABS®) can also be used with a preadenylated adaptor oligo.

Specifically, 20 μl of the RNase-treated beads can be added to a single PCR tube. 80 μl of ligase mix (5 μl T4 RNA ligase 1 (NEW ENGLAND BIOLABS®), 10 μl 10×T4 RNA ligase buffer, 5 μl BC_0047 oligo at 50 μM, 50 μl 50% PEG 8000, and 10 μl 10 mM ATP) can be added to the 20 μl of beads in the PCR tube. 50 μl of the ligase mixed with the beads can be transferred into a new PCR tube to prevent too many beads from settling to the bottom of a single tube and the sample can be incubated at 25° C. for 16 hours.

US11427856B2. Methods and kits for labeling cellular molecules (2022-08-30). University of Washington [US]. Inventors: Georg Seelig [US], Richard Muscat [GB], Alexander B. Rosenberg [US].

+ Open protocol

+ Expand

9

Second Ligation Optimization Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols

Beads were resuspended into 50 µL of a ligation master mix with the following components: 2 U/µL T4 RNA ligase I (NEB), 1x NEB T4 RNA ligase I buffer, 2 µM second ligation oligo (Supplementary Data 5), 25% PEG 8000, 50 µM ATP, 7.5% DMSO, and 1 mM hexaammine cobalt chloride. After incubation at room temperature overnight (12+ h), the reaction was diluted with 50 µL water to reduce viscosity, washed once with high salt wash buffer and once with low salt wash buffer, and then resuspended in water. The amount of water was 6 µL per sample in the first ligation reaction, before pooling the barcoded samples. For example, if the second ligation mixture contains a pool of six samples, the amount of water used for resuspension would be 36 µL. Samples can be stored at 4 °C or frozen at −20 °C; both can be used for the next PCR step.

Watkins C.P., Zhang W., Wylder A.C., Katanski C.D, & Pan T. (2022). A multiplex platform for small RNA sequencing elucidates multifaceted tRNA stress response and translational regulation. Nature Communications, 13, 2491.

+ Open protocol

+ Expand

10

3'-End Labeling of RNA with [32P]-pCp

Check if the same lab product or an alternative is used in the 5 most similar protocols

RNA (10–20 pmol), 3-μl 10X T4 RNA ligase I buffer, 3-μl dimethyl sulfoxide (DMSO), 0.5-μl adenosine triphosphate (ATP) (75 mM), 2-μl T4 RNA ligase I (New England Biolabs) and 5-μl cytidine-3′,5′-bis(phosphate), [5′-³²P] (pCp, [5′-³²P]) (Perkin Elmer) were mixed to a final volume of 30 μl in an Eppendorf tube. The ligation reaction was carried out overnight at 4°C. The enzyme was removed by phenol/chloroform extraction. Labeled RNA was separated from unincorporated pCp, [5′-³²P] by passage through a NucAway spin column. Note that an additional nucleotide is added to the 3′ end of the RNA molecule in this labeling reaction, so the length of 3′-radiolabeled SRL RNA is 30 nt.

Ingle S., Azad R.N., Jain S.S, & Tullius T.D. (2014). Chemical probing of RNA with the hydroxyl radical at single-atom resolution. Nucleic Acids Research, 42(20), 12758-12767.

+ Open protocol

+ Expand

T4 rna ligase 1

Lab products found in correlation

Close spelling variants of this product

175 protocols using t4 rna ligase 1

cDNA Adapter Ligation for PCR Amplification

Mature miRNA Sequencing Protocol

Determining mRNA Terminal Sequences of traA Gene

Single-Stranded DNA Adapter Ligation for PCR

Single-Stranded DNA Adapter Ligation for PCR

Single-Stranded DNA Adapter Ligation for PCR

Single-Stranded DNA Adapter Ligation

3' cDNA Adapter Ligation

Second Ligation Optimization Protocol

3'-End Labeling of RNA with [32P]-pCp

About PubCompare

Ready to get started?