Blocking of the reporter was performed in a buffer containing 0.4 U/µl TdT, 4 mM ddCTP (GE healthcare), 1× TdT buffer (New England Biolabs), 0.25 mM CoCl2 (New England Biolabs, provided with TdT), nuclease-free water (New England Biolabs), and 10 µM reporter. The total reaction volume used was 50 µl. The mixtures were subsequently incubated at 37°C for 3 h and heat inactivated at 70°C for 15 min. Afterward the reactions were filtered with G25 columns (Roche) according to the manufacturer’s instructions. The total end concentration of the reporter was calculated via the dilution factor (end concentration = (original reaction volume (50 µl) × original concentration (20 µM))/end volume). Afterward the blocked reporters were checked for remaining 3′-OH ends by performing an elongation reaction with TdT and dTTP nucleotides. This reaction contained 1× TdT buffer (New England Biolabs), 0.25 mM CoCl2 (New England Biolabs, provided with TdT), 0.4 U/µl TdT, 4 mM dTTP (ThermoFisher), and 300 nM reporter. The final end volume was 50 µl. The samples were subsequently incubated at 37°C for 1 h and heat inactivated 15 min at 70°C.
Cocl2
Manufactured by New England Biolabs
Sourced in United States, Germany
CoCl2 is a chemical compound used as a laboratory reagent. It is a blue-colored crystalline solid that is soluble in water. CoCl2 is commonly used as a colorimetric indicator in various applications, such as detecting the presence of water in organic solvents.
Automatically generated - may contain errors
Lab products found in correlation
Tdt buffer, by New England Biolabs (4 mentions)
Terminal transferase, by New England Biolabs (3 mentions)
Protease, by Qiagen (1 mentions)
Taq dna polymerase, by Vazyme (1 mentions)
Quick cip enzyme, by New England Biolabs (1 mentions)
Cutsmart buffer, by New England Biolabs (1 mentions)
Biotin 16 dctp, by Jena Biosciences (1 mentions)
T4 dna polymerase, by New England Biolabs (1 mentions)
Lbcas12a, by New England Biolabs (1 mentions)
Nuclease free water, by New England Biolabs (1 mentions)
Ddctp, by GE Healthcare (1 mentions)
3 biotin labeled oligonucleotides, by Integrated DNA Technologies (1 mentions)
Biotin dutp, by Roche (1 mentions)
8 protocols using cocl2
1
Terminal Deoxynucleotidyl Transferase-Mediated Reporter Blocking
2
DNA Inactivation and Fragmentation
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To inactivate the 3’-hydroxyl terminus of DNA molecules, a single ddGMP was added to the ends of 2 μg of amplified cDNA in a 50 μL mixture containing 0.1 mM ddGTP (Roche, 03732738001), 0.25 mM CoCl2 (NEB, M0315), 20 U of Terminal Transferase (NEB, M0315) and 1× TdT buffer (NEB, M0315). The reaction was performed at 37 °C for 2 h and stopped by heating for 20 min at 75 °C. Thereafter, the DNA sample was purified using AMPure XP beads and eluted in 24 μL of nuclease-free water. Three microliters of Quick CIP enzyme (NEB, M0525) and 3 μL of 10× Cutsmart buffer (NEB, B7204) were added to the eluate, and the mixture was incubated at 37 °C for 30 min for dephosphorylation. After inactivation of the phosphatase at 80 °C for 5 min, the DNA molecules were cut with 10 pmol of Cas9-gRNA complex (2.5 pmol each) at 37 °C for 15 min. For the removal of Cas9 protein from cleavage sites, the sample was digested with 1.5 μL of protease (Qiagen, 19155) for 10 min at 56 °C, followed by 70 °C for 15 min to inactivate protease. dA-tailing was carried out at 72 °C for 5 min with 1 μL of 10 mM dATP (NEB, N0440) and 5 U of Taq DNA polymerase (Vazyme, P101-d1-AC). The product was used directly for adapter ligation through SQK-LSK110 (ONT) or barcoding through NBD114 (ONT).
3
Terminal Transferase Mediated Slat Elongation
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Terminal transferase (TdT) was used to synthesize a poly-T brush on the 3′-end of each v8 slat. TdT was used here because slats as-purchased were 90 nt and further increasing their length for the brush would have resulted in a significant upcharge from the commercial vendor. The TdT poly-T brush reaction were as follows: PAGE purified x- and y-slats were separately combined with dTTPs (Fisher Scientific, R0171) at a molar ratio of 1:125, 0.05 v/v% terminal transferase (NEB, M0315L) in 1× terminal transferase reaction buffer (NEB, M0315L) and 0.25 mM CoCl2 (NEB, M0315L). The reaction was incubated for 30 min at 37 °C, followed by heat inactivation of the enzyme at 70 °C for 10 min. The slats were then separated from the reaction buffer by precipitating them in isopropanol, washed twice in cold ethanol, and resuspending them in the water at the desired concentration. The DNA slat assembly reactions were then prepared as described above.
4
Oligo-based Viral Detection Labeling
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PAGE-purified Oligo was ordered from a commercial provider and diluted to 10 μM before use (Table S1 ). To assemble the labeling reaction, 1 μL probe oligo (usually, we utilize two probes for one virus detection), 1 μL dATP of 1 mM (NEB, Ipswich, MA, USA), 1 μL biotin-16-dCTP of 1 mM (Jena Bioscience, Jena, Thuringia, Germany), 1 μL T4 DNA polymerase (NEB, Ipswich, MA, USA), 1 μL 10× T4 DNA polymerase buffer (NEB, Ipswich, MA, USA), and DEPC water were added to make a 10 μL reaction. The reaction was incubated at 25 °C for 15 min and then at 37 °C for 1 h. The entire reaction was added to the hybridization tube during the hybridization step. For terminal deoxynucleotidyl transferase reaction, 1 μL 10× TdT buffer (NEB, Ipswich, MA, USA), 1 μL CoCl2 of 2.5 mM (NEB, Ipswich, MA, USA), 1 μL probe oligo, 1 μL biotin-16-dCTP of 1 mM (Jena Bioscience, Jena, Thuringia, Germany), 1 μL Terminal nucleotide Transferase (NEB, Ipswich, MA, USA), and DEPC water were added to make a 10 μL reaction. The reaction was incubated at 37 °C for 1 h.
5
Cas12a-Mediated Fluorescent Labeling
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Cas12a activation and TdT elongation were performed in one step containing 50 nM LbCas12a (New England Biolabs), 62.5 nM crRNA2, 300 nM blocked reporter (as indicated in text, e.g. F20C), plasmid (concentration indicated in figures), 1× TdT buffer, 0.25 mM CoCl2 (New England Biolabs), 0.4 U/µl TdT (New England Biolabs), 4 mM dTTP, and F-dUTP (1 μM) or B-dUTP (1 μM) (as indicated). The final volume was 100 µl. The samples were subsequently incubated at 25° or 37°C for 30 min and heat inactivated by incubation at 70°C for 15 min or via addition of 20 mM EDTA final concentration before adding to the lateral flow stick.
6
Exome Capture and Circularization Protocol
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The list of the entire exons for the 30 genes was obtained from a consensus coding sequence database, CCDS (build 36.3), showing a total number of 701 exons covering 102 kb, according to hg18 (March, 2006 assembly).
Targeted restriction fragments were selected using Disperse software [11 (link)]. Templates for circularization of each chosen targeted fragment (selector probes) were designed using ProbeMaker software [12 (link)]. Each selector probe consisted of two sequences of 20-25 nucleotides complementary to the ends of its targeted restriction fragment.
The 3'-biotin-labeled oligonucleotides (Integrated DNA Technologies, Coralville, IA, USA) were prepared by incubating the oligonucleotides with 1× Tdt buffer (NEB), 1× CoCl2 (NEB), 0.1 mM dUTP-biotin (Roche Diagnostics, Mannheim, Germany), and 0.2 units/µL terminal transferase (NEB) in a final volume of 50 µL. The reaction was incubated at 37℃ for 1 h and followed by enzyme inactivation at 75℃ for 20 min.
Targeted restriction fragments were selected using Disperse software [11 (link)]. Templates for circularization of each chosen targeted fragment (selector probes) were designed using ProbeMaker software [12 (link)]. Each selector probe consisted of two sequences of 20-25 nucleotides complementary to the ends of its targeted restriction fragment.
The 3'-biotin-labeled oligonucleotides (Integrated DNA Technologies, Coralville, IA, USA) were prepared by incubating the oligonucleotides with 1× Tdt buffer (NEB), 1× CoCl2 (NEB), 0.1 mM dUTP-biotin (Roche Diagnostics, Mannheim, Germany), and 0.2 units/µL terminal transferase (NEB) in a final volume of 50 µL. The reaction was incubated at 37℃ for 1 h and followed by enzyme inactivation at 75℃ for 20 min.
7
TUNEL Assay for Apoptosis Detection
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TUNEL was performed as previously described [42 (link)] with the following modifications. Worms were killed, fixed, and formamide-bleached as described in the in situ hybridization section. Worms were then incubated for four hours at 37°C in 20 μL of TdT reaction mix (0.8 μM DIG-dUTP, 39.2 μM dATP, 1× reaction buffer (New England Biolabs #M0315L), 250 μM CoCl2 (New England Biolabs #M0315L), 0.5 units/μL terminal transferase (New England Biolabs #M0315L)–final concentrations). Worms were then washed, blocked, and incubated overnight with anti-DIG-POD (peroxidase) (1:2000, Millipore-Sigma #11207733910) as described in the in situ hybridization section.
8
Terminal Deoxynucleotidyl Transferase-Mediated 3'-End Elongation
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Elongation of the 3′-hydroxyl (3′-OH) ends was started by the addition of TdT to the products generated in the Cas12a activation step. The 50 µl of added volume contained 1 × TdT buffer (New England Biolabs), 0.5 mM CoCl2 (New England Biolabs, provided with TdT), 0.8 U/µl TdT and 8 mM dTTP. The final concentrations in the 100 µl total volume of all the relevant components were therefore 1 × TdT buffer, 0.25 mM CoCl2, 0.4 U/µl TdT and 4 mM dTTP. The samples were subsequently incubated at 37°C for 3 h.
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