For 5’ modification and PS bond modification: primers with 2PS bonds, Acrydite™, C6-Alex 647, amine C6 and amine C12 were ordered from IDT. Bis-PEG10-NHS ester was purchased from Broadpharm (BP-22588). Biotin NHS ester (H1759), azidoacetic acid NHS ester (900919), s-acetylthioglycolic acid NHS ester (A9043), and propargyl NHS ester (764221) were purchased from Sigma-Aldrich. NHS esters were incubated with 10 μM primers with 5’ amine C6 or C12 group at 1 mM concentration in 1 x borate buffer (Thermo Fisher, 28341) overnight at room temperature. Primers were then desalted using Bio-Spin 30 columns (Bio-rad). The labelling efficiencies were measured by HPLC and MALDI-TOF mass spectrometry (Supplementary Fig. 3 ). For 3’ Am-ddU modification: An aliquot of 10 μg dsDNA donors, 20 μM amino-11-ddUTP (Lumiprobe), 50 μM CoCl2, and 1 U TdT polymerase (New England Biolabs), 1 x TdT reaction buffer were incubated in a 50 μl reaction at 37 °C for 4 h. amino-11-ddUTP modified dsDNA was purified using QlAquick PCR purification kit (Qiagen) after stopping the reaction with 10 μl 0.2 M EDTA (PH = 8.0). The resulting product was then used to generate 3’ ddU-PEG10 modified dsDNA by incubation with Bis-PEG10-NHS ester at conditions described above.
Amino 11 ddutp
Manufactured by Lumiprobe
Amino-11-ddUTP is a modified nucleotide, specifically a dideoxyuridine triphosphate derivative with an amino group attached to the 11th carbon position. It is a versatile biochemical tool used in various molecular biology applications.
Automatically generated - may contain errors
Lab products found in correlation
Borate buffer, by Thermo Fisher Scientific (2 mentions)
Bio spin 30 columns, by Bio-Rad (2 mentions)
Qlaquick pcr purification kit, by Qiagen (2 mentions)
Biotin nhs ester h1759, by Merck Group (2 mentions)
Azidoacetic acid nhs ester 900919, by Merck Group (2 mentions)
S acetylthioglycolic acid nhs ester a9043, by Merck Group (2 mentions)
Propargyl nhs ester, by Merck Group (2 mentions)
Tdt polymerase, by New England Biolabs (2 mentions)
G 25 spin columns, by GE Healthcare (1 mentions)
Terminal deoxynucleotidyl transferase, by New England Biolabs (1 mentions)
Stellaris probe designer, by Biosearch Technologies (1 mentions)
5 protocols using amino 11 ddutp
1
Efficient Primer and DNA Modifications
2
Efficient Primer and DNA Modifications
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For 5’ modification and PS bond modification: primers with 2PS bonds, Acrydite™, C6-Alex 647, amine C6 and amine C12 were ordered from IDT. Bis-PEG10-NHS ester was purchased from Broadpharm (BP-22588). Biotin NHS ester (H1759), azidoacetic acid NHS ester (900919), s-acetylthioglycolic acid NHS ester (A9043), and propargyl NHS ester (764221) were purchased from Sigma-Aldrich. NHS esters were incubated with 10 μM primers with 5’ amine C6 or C12 group at 1 mM concentration in 1 x borate buffer (Thermo Fisher, 28341) overnight at room temperature. Primers were then desalted using Bio-Spin 30 columns (Bio-rad). The labelling efficiencies were measured by HPLC and MALDI-TOF mass spectrometry (Supplementary Fig. 3 ). For 3’ Am-ddU modification: An aliquot of 10 μg dsDNA donors, 20 μM amino-11-ddUTP (Lumiprobe), 50 μM CoCl2, and 1 U TdT polymerase (New England Biolabs), 1 x TdT reaction buffer were incubated in a 50 μl reaction at 37 °C for 4 h. amino-11-ddUTP modified dsDNA was purified using QlAquick PCR purification kit (Qiagen) after stopping the reaction with 10 μl 0.2 M EDTA (PH = 8.0). The resulting product was then used to generate 3’ ddU-PEG10 modified dsDNA by incubation with Bis-PEG10-NHS ester at conditions described above.
3
GOLD FISH Probe Labeling Protocol
4
Single-molecule FISH for CVB3 RNA
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Single-molecule Fluorescence In Situ Hybridization (smFISH) was performed as described previously (Lyubimova et al., 2013 (link); Raj et al., 2008 (link)). Custom-made oligonucleotide probes (96) targeting the positive RNA strand of CVB3 were designed using the website https://www.biosearchtech.com/ (See Table S2 for sequence of the probes). Probes were labeled with Atto633-NHS (Atto-Tec), as described previously (Gaspar et al., 2018 ). In brief, Atto633-NHS was dissolved in DMSO and mixed with NaHCO3 (final concentration 0.05 M; pH 8.4) and Amino-11-ddUTP (5 mM; Lumiprobe). The oligonucleotides were labeled by mixing 200 μM of each oligo with Terminal deoxynucleotidyl Transferase (TdT) buffer, 10 mM dye solution, and TdT (ThermoFischer) and incubating at 37þC overnight. Next, the labeled probes were precipitated using 100% ethanol, washed with 80% ethanol to remove free dye, and resuspended in nuclease-free water to a final concentration of 30 μM. Prior to hybridization, probes were diluted in TE to a working stock concentration of 1 μM.
5
smFISH Probe Design and Preparation
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smFISH Probe Design and Preparation smFISH oligonucleotide probes were designed using Stellaris Probe Designer (Biosearch Technologies). Probes sets contain between 45 and 48 non-overlapping 20-nucleotide oligos. A full list of all probe sets is provided in Supplementary file 1 . Anti-GFP probes were prepared by conjugating NHS-ester ATTO 633 dye (Sigma 01464) to the 3' end of each oligonucleotide. Anti-Sens probes were prepared by conjugating NHS-ester ATTO 565 dye (Sigma 72464) to the 3' end of each oligonucleotide. These oligos bear a mdC(TEG-Amino) 3ʹ modification to allow conjugation, and were obtained from Biosearch Technologies. Conjugation and purification was performed as described (Little and Gregor, 2018 (link)). All other probe sets were prepared using the enzymatic conjugation protocol as described (Gaspar et al., 2017 (link)). Briefly, amino-11-ddUTP (Lumiprobe) was conjugated to NHS-ester ATTO 633. Terminal deoxynucleotidyl transferase (New England Biolabs) was then used to conjugate ATTO 633-ddUTP to the 3' ends of oligonucleotides that had been purchased from IDT. After enzymatic conjugation, oligos were purified from free ATTO 633-ddUTP using G-25 spin columns (GE Illustra) according to manufacturer’s instructions. Final concentration of oligonucleotide was 33 µM in water. Probes were stored at −20°C, protected from light, until use.
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