Each multiplex ligation mix included 5 nM individual MOLigo pair probes (Table 2 ) with 2.5 μL10X Hifi Taq DNA ligase reaction buffer, 0.5 μL Hifi Taq DNA ligase (New England BioLabs, Massachusetts, USA), 0.1 μL of 0.05 ng/μL IC cloned into the plasmid and 2.5 μL template DNA. The reaction was brought to a final volume of 25 μL with H2O. Ligation protocol in the thermocycler (DNA Engine Dyad, Bio-Rad, Foster City, CA, USA) was set to: 10 min of denaturation at 95 °C followed by 20 cycles of 30 s at 95 °C and 1 min at 59 °C. Ligation products were stored at 10 °C until the next step of the MOL-PCR reaction.
Hifi taq dna ligase
Manufactured by New England Biolabs
Sourced in United States
Hifi Taq DNA ligase is a high-fidelity DNA ligase enzyme that catalyzes the formation of phosphodiester bonds between juxtaposed 5' phosphate and 3' hydroxyl termini in double-stranded DNA. It exhibits robust ligation activity and high specificity for blunt and cohesive DNA ends.
Automatically generated - may contain errors
Lab products found in correlation
Dna engine dyad, by Bio-Rad (1 mentions)
Polyjet dna transfection reagent, by SignaGen (1 mentions)
T4 pnk kinase, by New England Biolabs (1 mentions)
T100 thermal cycler, by Bio-Rad (1 mentions)
Hifi taq dna ligase reaction buffer, by New England Biolabs (1 mentions)
Veriti 96 well thermal cycler, by Thermo Fisher Scientific (1 mentions)
Qubit dsdna br assay kit, by Thermo Fisher Scientific (1 mentions)
Adenosine triphosphate (atp), by New England Biolabs (1 mentions)
Taq dna ligase, by New England Biolabs (1 mentions)
T7 dna ligase, by New England Biolabs (1 mentions)
E coli dna ligase, by New England Biolabs (1 mentions)
Neb10β, by New England Biolabs (1 mentions)
Q5 dna polymerase, by New England Biolabs (1 mentions)
T4 polynucleotide kinase, by New England Biolabs (1 mentions)
T4 dna ligase, by New England Biolabs (1 mentions)
6 protocols using hifi taq dna ligase
1
Multiplex Ligation-Dependent Probe Amplification
2
Synthetic eccDNA Identification and Transfection
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Synthetic eccDNA sequences were identified by UCSC Genome Browser database. The linearized dsDNAs were acquired through PCR reaction and a PCR Purification Kit. The primer sequences for PCR of each genomic regions in SLC7A1 are listed in Supplementary Table 3 . Synthetic eccDNAs were prepared by circularizing the dsDNAs using HiFi Taq DNA ligase (NEB), and performed in thermocyclers with the following cycles: 95 °C for 3 min, 60 °C for 10 min and 37 °C for 5 min for at least 15 cycles. The synthetic eccDNAs were digested with Plasmid-Safe ATP-dependent DNase (Lucigen) and amplified by φ29 DNA polymerase (QIAGEN) before being regained with a PCR Purification Kit. Each junction site of the synthetic eccDNAs was verified through sequencing the PCR products by inverse primers. The inverse primer sequences for PCR of each genomic regions in SLC7A1 are listed in Supplementary Table 4 .
Synthetic eccDNAs transfection was carried out using PolyJet™ DNA Transfection Reagent (SignaGen, SL100688) according to the manufacturer’s instructions.
Synthetic eccDNAs transfection was carried out using PolyJet™ DNA Transfection Reagent (SignaGen, SL100688) according to the manufacturer’s instructions.
3
Ligase-Assisted Minicircle Accumulation (LAMA)
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The procedure of the LAMA method was previously reported [33 (link)]. Fragments A and E were purified with E.Z.N.A. Cycle-Pure Kit (Omega, Norcross, GA, USA) according to the manual to desalt and remove enzymes and contamination, and then phosphate groups were added to the 5′ end of these fragments by T4 PNK kinase (NEB) at 37 °C for 30 min following inactivation at 65 °C for 20 min. About 500 ng of fragment A and E were mixed in 5 µL 10× HIFI Taq DNA ligase buffer with 1 µL of HIFI Taq DNA ligase (NEB) for the Ligase-Assisted Minicircle Accumulation (LAMA) procedure. The mixture was placed in a thermal cycler (BIO-RAD T100 thermal cycler) that completes the following temperature program: step 1, DNA denaturation at 95 °C for 20 s; step 2, cooling at a maximum rate of 4 °C for 1 min; step 3, ligation at 65 °C for 20 min for 3–10 cycles. The DNA circles ranging from 700 bp to 3 kb were produced by this procedure.
4
High Molecular Weight DNA Digestion and Tagging
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High molecular weight genomic DNA (gDNA) was quantified using the Qubit dsDNA BR assay kit (Thermo Fisher Scientific, Cat.Q32850). A total of 40 μg of gDNA was incubated with 3μl of ClaI (NEB, Cat. R0197S) or AsiSI (NEB, Cat.R0630L) or PmeI (NEB, Cat.R0560L), or BamHI (NEB, Cat.R0136M) for 2 hours at 37°C, with gentle flicking every 20 mins. Subsequently, the enzyme was heat-inactivated at 65°C for 20 mins. Ligations were carried out using 4 μg of DNA per 50 μl reaction.
Tagging reactions were done in 50 μL volume for each reaction in a MicroAmp™ TriFlex Well PCR Reaction Plate (Applied Biosystems, Cat. A32811), with 4μl/reaction of 0.3μM duplex TeloTag adapter, 5μl/reaction of 10X HiFi Taq DNA Ligase Reaction Buffer (NEB, Cat. M0647S), and 1μl/reaction of HiFi Taq DNA Ligase (NEB, Cat. M0647S). The TeloTagging reactions were incubated for 5 mins at 65°C in a Veriti™ 96-Well Thermal Cycler (Applied Biosystems™, Cat.4375786). Ligations were done through 15 cycles of denaturing at 65°C for 1 min, followed by annealing and ligating at 45°C for 3 mins with a 15% ramp down of rate between steps.
Tagging reactions were done in 50 μL volume for each reaction in a MicroAmp™ TriFlex Well PCR Reaction Plate (Applied Biosystems, Cat. A32811), with 4μl/reaction of 0.3μM duplex TeloTag adapter, 5μl/reaction of 10X HiFi Taq DNA Ligase Reaction Buffer (NEB, Cat. M0647S), and 1μl/reaction of HiFi Taq DNA Ligase (NEB, Cat. M0647S). The TeloTagging reactions were incubated for 5 mins at 65°C in a Veriti™ 96-Well Thermal Cycler (Applied Biosystems™, Cat.4375786). Ligations were done through 15 cycles of denaturing at 65°C for 1 min, followed by annealing and ligating at 45°C for 3 mins with a 15% ramp down of rate between steps.
5
Cloning Experiments Using E. coli NEB10β
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E. coli strain NEB10β (New England Biolabs, MA) was used for all cloning experiments. Restriction enzymes, T4 polynucleotide kinase, T4 DNA ligase, T7 DNA ligase, E. coli DNA ligase, Taq DNA ligase, HiFi Taq DNA ligase, ATP, and Q5 DNA polymerase were purchased from New England Biolabs, MA. Plasmid-safe ATP-dependent DNase was obtained from Lucigen, WI. DNA purification buffers PB and PE were purchased from Qiagen, Germany. All DNA oligonucleotides were ordered from Integrated DNA Technologies (Coralville, IA). All DNA guides were ordered unphosphorylated and the phosphorylation reaction was performed by T4 PNK enzyme prior to DNA cleavage.
6
Variant-Specific SARS-CoV-2 Genotyping by OLA
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Following the pre‐amplification step, 10% (2 μl) of the RT‐PCR product was transferred into its SNP‐specific OLA reaction. Three SNP‐specific OLA reactions targeting E484/E484K, L452R, and G339D were employed for the detection of the four variant groups aforementioned. The RT‐PCR product encompassing AA339 was added to the G339D OLA reaction, and the product containing AA452 and AA484 was transferred to the L452R and E484/E484K OLA reactions. The ligation assay was conducted with the high‐fidelity, thermostable HiFi Taq DNA Ligase (NEB #M0647). Reactions were performed with 0.2 μl of the HiFi Taq Ligase and 2 μl of the 10× HiFi Taq Ligase Buffer. Three ligation probes, two VPs with different 3′ nucleotides, and one CP, specific for each SNP of interest, were added to their respective OLA reaction. The CP was added to have a final concentration of 1 nM. The VPs were added to have final concentrations of 10 nM, with the exception of the E484/E484K ligation set in which the VPs were at a final concentration of 1 nM to reduce cross‐over noise that was occurring in the reaction. This cross‐over reduction was observed by empirical study (data not shown). Molecular‐grade water was added to bring the reaction to 20 μl total. The reaction was performed with a 95°C hold for 1 min followed by a 60°C hold for 10 min. The ligation probe sequences are reported in Table 1 .
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