The reaction mix for circularization of the backbone and insert (3:1 backbone:insert ratio, 20 to 60 ng of insert), 5 μl 10× CutSmart Buffer (New England Biolabs), 10 μl 10 mM ATP (New England Biolabs), 2 μl T4 ligase (New England Biolabs), 2 μl SrfI (New England Biolabs), and MilliQ (until a volume of 50 μl)) was prepared on ice. Circularization was performed by 8 cycles of 10 min at 16 °C and 10 min at 37 °C, followed by 20 min at 70 °C. To digest any residual backbone-backbone byproducts, 1 μl of SrfI (New England Biolabs) was added and the mixture was incubated for 10 min at 37 °C, followed by 20 min at 70 °C. The linear DNA was then removed using Plasmid-Safe DNase (Lucigen). Briefly, the circularization mixture was combined with 6 μl 10× Plasmid-Safe Buffer (Lucigen), 2 μl Plasmid-Safe Enzyme (Lucigen), and 6 μl 10 mM ATP (New England Biolabs). Linear DNA was then digested by 30 min incubation at 37 °C, followed by 30 min inactivation at 70 °C. Circular DNA was purified using the QIAquick nucleotide removal kit according to the manufacturer’s protocol (Qiagen).
10 mm atp
Manufactured by New England Biolabs
10 mM ATP is a concentrated solution of adenosine triphosphate (ATP), a crucial energy-carrying molecule found in all living organisms. It functions as a cofactor for various enzymatic reactions involved in cellular processes such as energy production, signaling, and metabolism.
Automatically generated - may contain errors
Lab products found in correlation
Qiaquick nucleotide removal kit, by Qiagen (2 mentions)
T4 ligase, by New England Biolabs (2 mentions)
Cutsmart buffer, by New England Biolabs (2 mentions)
T4 polynucleotide kinase, by New England Biolabs (1 mentions)
5 mm dntps, by Jena Biosciences (1 mentions)
Superase in rnase inhibitor, by Thermo Fisher Scientific (1 mentions)
Poly a polymerase, by New England Biolabs (1 mentions)
Maxima h minus reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
4 protocols using 10 mm atp
1
Circular DNA Construction Protocol
2
Efficient Backbone Circularization Protocol
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The reaction mix for circularization of the backbone and insert (3:1 backbone:insert ratio, 20 to 60 ng of insert), 5 μl 10X CutSmart Buffer (New England Biolabs), 10 μl 10mM ATP (New England Biolabs), 2 μl T4 ligase (New England Biolabs), 2 μl SrfI (New England Biolabs), and MilliQ (until a volume of 50 μl)) was prepared on ice. Circularization was performed by 8 cycles of 10 minutes at 16°C and 10 minutes at 37°C, followed by 20 minutes at 70°C. To digest any residual backbone-backbone byproducts, 1 μl of SrfI (New England Biolabs) was added and the mixture was incubated for 10 minutes at 37°C, followed by 20 minutes at 70°C. The linear DNA was then removed using Plasmid-Safe DNase (Lucigen). Briefly, the circularization mixture was combined with 6 μl 10X Plasmid-Safe Buffer (Lucigen), 2 μl Plasmid-Safe Enzyme (Lucigen), and 6 μl 10mM ATP (New England Biolabs). Linear DNA was then digested by 30 minute incubation at 37°C, followed by 30 minutes inactivation at 70°C. Circular DNA was purified using the QIAquick nucleotide removal kit according to the manufacturer's protocol (Qiagen).
3
Oligonucleotide Phosphorylation and Annealing
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600 ng of oligonucleotides per annealing site (both, 3Cs h7SK- and hU6-oligonucleotides) were phosphorylated in two separate 20 μl reactions by mixing them with 2 μl 10× TM buffer (0.1 M MgCl2, 0.5 M Tris-HCl, pH 7.5), 2 μl 10 mM ATP (New England Biolabs), 1 μl 100 mM DTT (Cell Signaling Technology Europe), 20 units of T4 polynucleotide kinase (New England Biolabs) and water to a total volume of 20 μl. The mixture was incubated for 1 h at 37°C. Phosphorylated oligonucleotides were immediately annealed to purified multiplex dU-ssDNA template by adding both 20 μl phosphorylation products to 25 μl 10× TM buffer, 20 μg of dU-ssDNA template and water to a total volume of 250 μl. The mixture was denatured for 5 min at 95°C, annealed for 5 min at 55°C and cooled down for 10 min at room temperature.
4
Reverse Transcription of ncRNA and mRNA
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cDNA was generated in a total reaction volume of 20 μl using poly(A) tailing-based RT. The RT reaction mix was as follows: 4 μl RT-buffer (5X), 1 μl 2.5 μM poly A adapter/primer (Integrated DNA Technologies, Inc.), 1 μl 5 mM dNTPs (Jena Bioscience), 0.25 μl Maxima™ H Minus reverse transcriptase (Thermo Fisher Scientific, Inc.), 0.25 μl SUPERase In™ RNase inhibitor (Thermo Fisher Scientific, Inc.), 0.5 μl 10 mM ATP (New England BioLabs, Inc.), 0.25 μl poly A polymerase (New England BioLabs, Inc.) and the required amount of RNA sample. Briefly, 1,000 ng of isolated intracellular ncRNA and 50 ng of isolated microvesicular extracellular ncRNA were used for RT. Due to the small amount of ncRNA in serum and urine, a fixed volume of 5 μl was used. RT was performed at 42°C for 30 min and 85°C for 10 min. Processed cDNA was stored at 4°C. Additionally, 2,000 ng of RNA was used for the RT of mRNA. The RT reaction mix contained 5 μl RT-buffer (5X), 1 μl 5 mM dNTPs (Jena Bioscience), 1 μl RT primer (10 μM), 0.25 μl Maxima™ H Minus reverse transcriptase (Thermo Fisher Scientific, Inc.), 0.25 μl SUPERase In™ RNase inhibitor (Thermo Fisher Scientific, Inc.) and the RT was carried out in a total volume of 25 μl. The RT temperature protocol was performed as follows: 65°C for 1 min, 25°C for 10 min, 50°C for 45 min and 85°C for 10 min.
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