Biotinylated lambda DNA used in SWR1 sliding on naked DNA assays was purchased from LUMICKS (SKU: 00001). Lambda DNA used in nucleosome array assays was made with 3 biotins on one end, and 3 digoxigenin on the other end using the following protocol. Custom oligos were ordered from IDT with sequences listed in Table 1 (6–7). Lambda DNA was ordered from NEB (cat# N3011S). Oligo 1 was annealed to lambda DNA by adding a 25-fold molar excess of oligo to lambda DNA, in an annealing buffer containing 30 mM HEPES pH 7.5 and 100 mM KCl. This mixture was heated to 70°C for 10 min and allowed to cool slowly to room temperature on the benchtop. 2 µl of NEB T4 DNA ligase (400 U, cat# M0202S) was added along with T4 DNA ligase buffer containing ATP and allowed to incubate at room temperature for 30 min. Then 50-fold molar excess of oligo 2 was added to the mixture along with an additional 1 µl of T4 DNA ligase and T4 DNA ligase buffer (NEB) with ATP adjusting for the change in volume and allowed to incubate at room temperature for 30 min. The resulting mixture was heat inactivated at 65°C for 10 min. End-labeled lambda DNA was purified using Qiaex II gel-extraction DNA clean-up kit following the manufactures’ instructions (Qiagen cat# 20021).
T4 dna ligase
Manufactured by New England Biolabs
Sourced in United States, China, United Kingdom, Germany, Japan, France, Canada, Morocco, Switzerland, Australia
T4 DNA ligase is an enzyme that catalyzes the formation of phosphodiester bonds between adjacent 3'-hydroxyl and 5'-phosphate termini in DNA. It is commonly used in molecular biology for the joining of DNA fragments.
Automatically generated - may contain errors
Lab products found in correlation
Restriction enzyme, by New England Biolabs (140 mentions)
Restriction endonuclease, by New England Biolabs (115 mentions)
T4 polynucleotide kinase, by New England Biolabs (93 mentions)
Qiaquick gel extraction kit, by Qiagen (85 mentions)
Q5 high fidelity dna polymerase, by New England Biolabs (65 mentions)
Qiaprep spin miniprep kit, by Qiagen (65 mentions)
Phusion high fidelity dna polymerase, by New England Biolabs (58 mentions)
Qiaquick pcr purification kit, by Qiagen (57 mentions)
T4 dna ligase buffer, by New England Biolabs (54 mentions)
Xhoi, by New England Biolabs (51 mentions)
Ecori, by New England Biolabs (48 mentions)
Bamhi, by New England Biolabs (47 mentions)
Antarctic phosphatase, by New England Biolabs (45 mentions)
Phusion dna polymerase, by New England Biolabs (42 mentions)
Taq dna polymerase, by New England Biolabs (40 mentions)
Phusion polymerase, by New England Biolabs (39 mentions)
T4 pnk, by New England Biolabs (39 mentions)
Dpni, by New England Biolabs (35 mentions)
Q5 dna polymerase, by New England Biolabs (35 mentions)
T4 dna polymerase, by New England Biolabs (34 mentions)
2 059 protocols using t4 dna ligase
1
Biotinylated and Digoxigenin-Labeled Lambda DNA
2
XACTLY Ligation: Adaptive NGS Library Prep
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
XACTLY ligation consisted of an initial ligation step and a subsequent nick repair ligation step prior to standard NGS library amplification and indexing. First, 0.05 pmol of substrate DNA (control/NA12878/cfDNA) was combined with 1 pmol of XACTLY adapters in a 60 μl ligation reaction with 800 units of T4 DNA ligase (New England Biolabs) and 1× T4 DNA ligase Buffer, and incubated at 20°C for 1 h, followed by either a 2× AMPure clean for control oligos, or a 1.2× AMPure clean for NA12878 or cfDNA. After DNA purification, DNA was again phosphorylated with 20 units of T4 Polynucleotide Kinase (New England Biolabs) and 1× T4 DNA ligase buffer in a 48.8 μl reaction and incubated at 37°C. After 30 min, 480 units of T4 DNA ligase was added to the reaction and the temperature reduced to 20°C for 15 min. Nick repair was followed by a 2× AMPure bead clean and elution in 20 μl of low TE (10 mM Tris pH 8, 0.1 mM EDTA).
3
Plasmid Construction for sp_bub1 Mutants
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Plasmids used in this study were listed in Table S2 . Plasmids were constructed either by the one-step cloning technique based on homologous recombination (Vazyme Biotech) or by the traditional digestion–ligation cloning method with enzymes purchased from NEB. To generate plasmids bearing spbub1 mutations, a spbub1 promoter and the coding sequences of full-length spbub1 were obtained by PCR using yeast genomic DNA as a template. The PCR products were then digested with BglII and NotI and ligated into a pJK148 vector with a T4 DNA ligase (NEB). The Ser532 site of spbub1 was mutated into Ala or Asp by ClonExpress II One Step Cloning Kit (Vazyme Biotech). To generate pFastBac-Mph1-His (used for expression of Mph1-His in insect cells), mph1 was obtained by PCR using a yeast cDNA library as a template and digested with BssHII and NotI, followed by ligation with a T4 DNA ligase (NEB).
4
Cassava Barcoded Adapter Ligation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The ligation reaction was completed using NEB 1x T4 DNA Ligase Reaction Buffer with 50 mM Tris-HCl, 10 mM MgCl2, 10 mM DTT, and 1 mM ATP, and 100 U of T4 DNA Ligase (New England BioLabs Inc., Ipswich, MA, M0202) for each sample. For cassava, a 1.5-μM barcode adapter and 1.5-μM HpaII-methylation adapter/MspI-methylation adapter were added to the two sets of samples. The ligation reaction was incubated over night at 16°C, and then the ligase was inactivated prior to the pooling of the samples by incubation at 65°C for 20 min.
5
Generating Chimeric ASIC Mutants
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mouse cDNAs for ASIC1a, ASIC2a, and ASIC2b have been described previously (Kweon et al., 2015 (link), 2016 (link)). Chimeric ASIC mutants were generated using an
overlap extension PCR strategy (Lee et al.,
2010 ; Kweon et al., 2016 (link)).
PCR products were ligated into pEGFP-C1 (Clontech) using T4 DNA ligase (New
England Biolabs). In the concatemer of ASIC2a-Mut2-ASIC2a, individual subunits
are linked by one asparagine followed by seven successive residues of glutamine
(van Bemmelen et al., 2015 (link)). Point
mutations and deletion of target sequences in ASIC2a mutants were generated by
inverse PCR reactions with Pfu Turbo DNA polymerase (Agilent Technologies),
followed by digestion with DpnI (Agilent Technologies). The digested products
were phosphorylated at the 5′ end using T4 polynucleotide kinase
(Enzynomics) and ligated into pEGFP-C1 (Clontech) using T4 DNA ligase (New
England Biolabs). The primers used to generate the ASIC mutants and concatemer
are listed in Tables S1, S2, and S3. Mutations were confirmed by DNA
sequencing.
overlap extension PCR strategy (
2010
PCR products were ligated into pEGFP-C1 (Clontech) using T4 DNA ligase (New
England Biolabs). In the concatemer of ASIC2a-Mut2-ASIC2a, individual subunits
are linked by one asparagine followed by seven successive residues of glutamine
(van Bemmelen et al., 2015 (link)). Point
mutations and deletion of target sequences in ASIC2a mutants were generated by
inverse PCR reactions with Pfu Turbo DNA polymerase (Agilent Technologies),
followed by digestion with DpnI (Agilent Technologies). The digested products
were phosphorylated at the 5′ end using T4 polynucleotide kinase
(Enzynomics) and ligated into pEGFP-C1 (Clontech) using T4 DNA ligase (New
England Biolabs). The primers used to generate the ASIC mutants and concatemer
are listed in Tables S1, S2, and S3. Mutations were confirmed by DNA
sequencing.
6
Cloning hupB and hlp Genes into Expression Vectors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To clone hupB into pET28a (pNA1; Supplementray Table 2 ), using the genomic DNA of Mtb as the template, a primer pair, KAP403F and KAP404R (Supplementary Table 3 ), and a Phusion Taq polymerase (Thermo Fisher Scientific), we PCR-amplified hupB and digested the eluted (HiMedia) amplicon with NdeI (NEB, United States) and BamHI (NEB). The digested and purified amplicon was ligated [using T4 DNA ligase (NEB)] to similarly digested pET28a to obtain 6X-HishupB. The episomal expression vector pVV16 (Yaseen et al., 2018 (link)) containing hupB (pNA4; Supplementary Table 2 ) was from our lab collection. To clone hlp, using the genomic DNA of Msm as the template, a primer pair, KAP634F and KAP635R, (Supplementary Table 3 ), and a Phusion Taq polymerase (Thermo Fisher Scientific), we PCR-amplified hlp and digested the eluted (HiMedia) amplicon with EcoRI (NEB) and HindIII (NEB). The digested and purified amplicon was ligated [using T4 DNA ligase (NEB)] to similarly digested pVV16 to obtain pVV16+hlp (pNA5; Supplementary Table 2 ). The molecular construction of pNA2 and 3 is provided in the subsection “KO generation and its complementation” (refer below).
7
Biotinylated Lambda DNA Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Biotinylated primer was annealed to Lambda DNA (N3011, NEB). The annealing protocol was: 100 μl (500 ng/μl) Lambda DNA was mixed with 1 μM biotinylated primer, and then the sample was incubated at 65 °C for 5 min, and the temperature was slowly decreased to the room temperature for another 45 min. Then 10× T4 DNA ligase buffer and 5 μl T4 DNA ligase (M0202, NEB) were added into the mix. The mix was incubated at 42 °C overnight. After overnight incubation, Buffer A (30% PEG 8000 and 10 mM MgCl2) was added to dilute the mix. The volume of Buffer A was the half volume of the mix. The new mix was incubated at 4 °C with rotation for 1 day, and then centrifuged at 18,000 g for 5 min by a centrifuge (Lynx 4000, THERMO FISHER). Finally, the pellet including the biotinylated Lambda DNA was dissolved by 100 μl TE150 buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH 8.0), and 150 mM NaCl).
8
Proximity Ligation of Nuclei
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After dA tailing and reaction stop, washing twice with NicE-C wash buffer, the nuclei were resuspended in 600 μL 1× T4 DNA ligase buffer containing 30 μL of 10% Triton X-100, 30 μL of 50% PEG8000, 20 μL of 50 μM bridge linker, 4 μL of T4 DNA ligase (NEB M0202S). Then, the nuclei were incubated for 4 h at 23°C for proximity ligation with interval shaking (1100 rpm, 15 sec every 2 min).
9
Illumina and Nanopore RNA Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Illumina libraries were prepared using a TruSeq stranded total RNA kit (Illumina, USA) following the manufacturer’s instructions and sequenced on a HiSeq 3000 sequencer (Illumina).
A direct RNA sequencing library was prepared using a direct RNA sequencing kit (SQK-RNA001; Oxford Nanopore Technologies) from approximately 500 ng of poly(A) RNA input following the manufacturer’s instructions. Briefly, a reverse transcription adapter was ligated using T4 DNA ligase (New England Biolabs), and first-strand cDNA was synthesized using SuperScript III reverse transcriptase (Thermo Fisher Scientific). Sequencing adapters were ligated using T4 DNA ligase (New England Biolabs). The library was loaded onto a MinION flow cell and sequenced for 48 h. The RNA calibration strand (RCS) spike-in was added to the run in the first step as a control.
A direct RNA sequencing library was prepared using a direct RNA sequencing kit (SQK-RNA001; Oxford Nanopore Technologies) from approximately 500 ng of poly(A) RNA input following the manufacturer’s instructions. Briefly, a reverse transcription adapter was ligated using T4 DNA ligase (New England Biolabs), and first-strand cDNA was synthesized using SuperScript III reverse transcriptase (Thermo Fisher Scientific). Sequencing adapters were ligated using T4 DNA ligase (New England Biolabs). The library was loaded onto a MinION flow cell and sequenced for 48 h. The RNA calibration strand (RCS) spike-in was added to the run in the first step as a control.
10
Construction of CRISPR Plasmid Library
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All plasmids and oligonucleotide primers for polymerase chain reaction (PCR) amplification used in this work are listed in Supplementary Tables S2 and S3 , respectively. Bacterial genomic or plasmid DNA was prepared using standard procedures [44 ]. The gold gate cloning method was used to generate a specific CRISPR array in plasmid sgRNA-bacteria as previously described [46 (link),47 (link)]. The two newly synthesized complementary oligonucleotide fragments were annealed in T4 oligo DNA annealing buffer (10 mmol/L Tris · pH 8.0, 50 mmol/L NaCl, 1 mmol/L EDTA). A 20 μL reaction system containing 300 ng of donor plasmid DNA, 10 pmol of annealed spacer DNA, 10 U of Bsa I, 10 U of T4 polynucleotide kinase and 200 U of T4 DNA ligase (all these enzymes were purchased from New England Biolabs) in T4 DNA ligase buffer was carried out for 60 cycles at 37 °C for 5 min and 16 °C for 5 min, followed by inactivation manipulation at 80 °C for 5 min. The ligation product was then directly transformed into recipient E. coli Mach1-T1 using previously described procedures [48 (link)].
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!