69kDa (Reducing)
10 mM Tris-HCl、50 mM KCl、1 mM DTT、0.1 mM EDTA、50% Glycerol pH 7.4 @ 25°C
Store at -25 ~ -15℃ for 2 years
[1] England, Thomas E , and O. C. Uhlenbeck . "3|[prime]|-Terminal labelling of RNA with T4 RNA ligase." Nature 275.5680(1978):560-1.
[2] Tessier, Daniel C. , R. Brousseau , and T. Vernet . "Ligation of single-stranded oligodeoxyribonucleotides by T4 RNA ligase." Analytical Biochemistry 158.1(1986):171-178.
T4 RNA Ligase 1 is an ATP-dependent enzyme that catalyzes the formation of phosphodiester bonds between the 5'-P and 3'-OH ends of single-stranded RNA, single-stranded DNA, or single nucleotides, either intermolecular or intramolecular. It is suitable for the connection of single-stranded RNA molecules and the 5' end junction constructed by miRNA libraries in NGS. Labeled with RNA 3 'terminal; Cyclization of oligonucleotides; tRNA modification; In 5'-RACE (Rapid Amplification of cDNA Ends) assay, it is used for oligonucleotides to bind to single-stranded cDNA; and the introduction of unnatural amino acids into proteins.
Name | Storage Temperature | quantity | concentration |
T4 RNA Ligase 1 | -20 ℃ | 100 ul or 500 µl | 10 U/µl |
10*Reaction Buffer | -20 ℃ | 1 ml | 10 X |
Adenosine 5'-Triphosphate (ATP) | -20 ℃ | 1 ml | 10 mM |
PEG8000 (RNase free) | -20 ℃ | 1 ml | 50% |
1. Set up a 20 μl reaction as follows:
Reagent | Volume | Final Concentration |
10 * Reaction Buffer | 2 ul | 1 X |
Single-stranded RNA with 5´P and 3´OH ends (200 ng-1 µg) | 1 ul | 10-50 ng/ul |
50% PEG8000 | 4 ul | 10% |
1mM ATP | 1 ul | 50 µM |
40 μ/ul Rnase inhibitor | 0.5ul | 1 U/µl |
10 U/µl T4 RNA Ligase 1 | 1ul | 0.5 U/µl |
DEPC-treated Water | Up to 20ul | - |
2. Incubate at 37°C for 1-2 hours.
For longer oligos, overnight incubation at 16°C for 16h may improve yield.
3. Incubate at 65°C for 15min to terminate the reaction.
Please avoid repeated freeze-thaw cycles.
In the experimental design, 133nt single-stranded RNA obtained by transcription in vitro was used. The 5 'end triphosphate group of RNA was removed and digested by RppH enzyme to produce 5' monophosphate group RNA, and then the RNA was connected by T4 RNA Ligase 1. This product was added to the 20µl reaction system, incubated at 37℃ for 120min for reaction, and incubated at 65℃ for 15min to inactivate the enzyme. Remove 9μl, add 1μl 10X RNA Loading Buffer, then perform 20% non-denatured polyacrylamide gel electrophoresis, and then stain with Gelred at room temperature for 20 min, and observe the experimental results under UV lamp. As shown in the figure, this product has the following effects.
Lane 1 Negative Control-1 (negative control with no added enzyme only);
Lane 2 Negative Control-2 (only negative controls without T4 RNA Ligase 1);
Lane 3 UA-T4 RNA Ligase 1 2U;
Lane 4 UA-T4 RNA Ligase 1 5U;
Lane 5 UA-T4 RNA Ligase 1 10U;
Lane 5 UA-T4 RNA Ligase 1 20U;
2μg (R: reducing condition, N: non-reducing condition).
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