DNA templates for IVT were amplified by PCR using the Q5 High Fidelity DNA Polymerase from New England Biolabs (NEB) (NEB #M0491S). Primers for PCR can be found in Table S4 . PCR product was checked by gel electrophoresis for specificity and then purified using the Zymo DNA Clean-up and Concentration kit (Zymo #D4033). DNA was eluted with nuclease-free water and stored at -20°C until use. IVT reactions were set up with the HiScribe™ SP6 RNA Synthesis Kit (NEB #E2070S) or HiScribe™ T7 High Yield RNA Synthesis Kit (NEB #E2040S). Reactions were prepared in a volume of 25 μL following manufacturer's protocol. To generate Z-mRNA or m1ψ-mRNA by IVT, 1.25 μL of 100 mM ZTP (TriLink Biotechnologies N-1001, 2-Amino-ATP) or 1.25 μL of 100 mM m1ψTP (TriLink Biotechnologies N-1081) was used to replace ATP or UTP, respectively. Reactions were incubated at 37°C for 2-4 hours. After incubation, 25 μL of nuclease-free water was added to each reaction along with 2 μL of DNase I (NEB #M0303A), and the reaction was incubated at 37°C for 15 minutes. Reactions were then purified using the Monarch RNA Clean-up Kit (NEB #T2040L) following manufacturer’s protocol. mRNA was eluted with nuclease free water and the concentration was determined by the Thermo Scientific NanoDrop 2000/2000c. mRNAs were stored at -80°C until use.
2 amino atp
Manufactured by TriLink
2-Amino-ATP is a nucleotide analog that serves as a chemical building block for various biochemical applications. It consists of an adenine base, a ribose sugar, and a triphosphate group. The 2-amino modification on the adenine ring distinguishes it from the natural ATP molecule. This product is intended for research use only.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000 2000c, by Thermo Fisher Scientific (2 mentions)
N 1001, by TriLink (2 mentions)
Dnase 1, by New England Biolabs (2 mentions)
Hiscribe sp6 rna synthesis kit, by New England Biolabs (1 mentions)
Hiscribe t7 high yield rna synthesis kit, by New England Biolabs (1 mentions)
Monarch rna cleanup kit, by New England Biolabs (1 mentions)
Q5 high fidelity dna polymerase, by New England Biolabs (1 mentions)
Zymo dna cleanup and concentration kit, by Zymo Research (1 mentions)
Precision grna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Rna clean concentrator 5 kit, by Zymo Research (1 mentions)
Qubit 4 fluorometer, by Thermo Fisher Scientific (1 mentions)
2 o methyl atp, by TriLink (1 mentions)
3 datp, by TriLink (1 mentions)
Tnp atp, by Thermo Fisher Scientific (1 mentions)
Ara atp, by TriLink (1 mentions)
3 protocols using 2 amino atp
1
Protocol for In Vitro Transcription of Modified mRNA
2
Synthesis of Cas12a crRNA and Cas9 sgRNA
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For Cas12a crRNA synthesis, DNA template oligos (contain SP6 promoter sequence, Supplementary Data File 1) for IVT were ordered from IDT (Integrated DNA Technologies, Inc.). 5 μM complementary oligos were annealed in 1 × NEB buffer 3.0 (New England Biolabs, Inc.) using the following program: 98 °C 5 min, 0.1 °C/s cool down to 4 °C. HiScribeTM SP6 RNA Synthesis Kit (NEB, E2070S) was used for IVT reactions according to the manufacturer’s protocol. To generate Z-incorporated crRNA, ZTP (TriLink Biotechnologies, N-1001, 2-Amino-ATP) was used to replace ATP in the reaction (Fig. S1a ). Reactions were incubated at 37 °C for 10 h to reach higher concentration. For Cas9 sgRNA synthesis, Precision gRNA Synthesis Kit (Thermo Scientific, A29377) was used to create the template DNA for IVT. DNA oligos were ordered from IDT as per kit’s instruction. To remove DNA template, 25 μL of nuclease-free water with 2 μL of DNase I (NEB, M0303S) were added to each 25 μL reaction, and the reactions were incubated at 37 °C for 15 min. Synthesized RNAs were purified using RNA Clean & Concentrator-5 kit (Zymo, R1016), and the concentrations were quantified by the NanoDrop 2000/2000c (Thermo Scientific) and Qubit 4 fluorometer. RNAs were stored at −80 °C until use.
3
Evaluating Modified Nucleotide Incorporation
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Modified nucleotides, 3′-dATP (Cordycepin), 2′-amino-ATP, 2′-azido-ATP, araATP and 2′-O-methyl-ATP were from Trilink Biotechnologies, TNP–ATP was from Invitrogen, and LNA–ATP was from Jena Biosciences. The 50mer DNA primer/ 67mer DNA template was created as described above. For each modified nucleotide incorporation reaction, a 100 μL reaction mix was prepared containing 1× ThermoPol Buffer, 10 nM primer-template, 25 nM wild-type WT PolD or PolD H931A and 1 μM modified nucleotide. Reactions were incubated for 0, 1 or 15 min at 65°C and 10 μl aliquots were removed and mixed with 50 mM EDTA to quench the reaction. Reaction products were analyzed by capillary electrophoresis as described above. The concentration of product (51 nt DNA with a FAM label) was graphed for each nucleotide. All assays were performed in triplicate to ensure experiment reproducibility.
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