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 | | | | | T4 RNA Ligase 2, truncated K227Q | | | New | |
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Description:
T4 RNA Ligase 2, truncated K227Q (T4 Rnl2tr K227Q) specifically ligates the pre-adenylated 5´ end of DNA or RNA to the 3´ OH end of RNA. The enzyme does not use ATP for ligation but requires pre-adenylated linkers.
T4 Rnl2tr K227Q is a point mutant of T4 RNA Ligase 2, truncated (NEB #M0242). Mutation of K227 in T4 RNA Ligase 2 reduces enzyme lysyl adenylation (1). This mutation further reduces the formation of undesired ligation products (concatemers and circles) by T4 Rnl2tr (2), possibly by reducing the trace activity of T4 Rnl2tr in transfer of adenylyl groups from linkers to the 5´-phosphates of input RNAs.
The exclusion of ATP, use of pre-adenylated linkers, and the reduced enzyme lysyl adenylation activity provide the lowest possible background in ligation reactions. This enzyme has been used for optimized linker ligation for the cloning of microRNAs (2).
Source:
T4 Rnl2tr K227Q is expressed as an MBP fusion from a plasmid in E. coli which encodes the first 249 amino acids of the full length T4 RNA Ligase 2 with a lysine to glutamine mutation at position 227.
Applications:- Ligate a pre-adenylated DNA or RNA sequence tag to any RNA 3´-end
- Join a single stranded adenylated primer to small RNAs for cDNA library creation
- Join a single stranded adenylated primer to RNA for strand-specific cDNA library construction
Reagents Supplied:
T4 Rnl2 truncated Reaction Buffer (10X)
T4 Rnl2tr K227Q Reaction Buffer
Enzyme Properties
Heat Inactivation:
65°C for 20 minutes
Specific Activity:
200,000 units/mg
Reaction & Storage Conditions
Reaction Conditions:
1X T4 Rnl2tr K227Q Reaction Buffer
Incubate at 25°C.
1X T4 Rnl2tr K227Q Reaction Buffer:
50 mM Tris-HCl
10 mM MgCl2
1 mM DTT
pH 7.5 @ 25°C
Unit Definition:
200 units is defined as the amount of enzyme required to give 80% ligation of a 31-mer RNA to the pre-adenylated end of a 17-mer DNA [universal miRNA cloning linker (NEB #S1315)] in a total reaction volume of 10 µl in 1 hour at 25°C.
5´-FAM- rArGrUrCrGrUrArGrCrCrUrUrUrArUrCrCrGrArGrArUrUrCrArGrCrArArUrA-3´
5´-rAppCTGTAGGCACCATCAAT–NH2-3´
Unit Assay Conditions:
1X T4 Rnl2tr K227Q Reaction Buffer supplemented to 10% (w/v) PEG MW 4000, 5 pmol of 5´-FAM labeled RNA, and 10 pmol preadenylated DNA linker. After incubation at 25°C for 1 hour, the ligated product is detected on a 15% denaturing polyacrylamide gel.
Molecular Weight:
71,406.97 daltons
Molarity:
14 µM
Concentration:
200,000 units/ml
Storage Conditions:
10 mM Tris-HCl
100 mM NaCl
0.1 mM Dithiothreitol
0.1 mM EDTA
50% Glycerol
pH 7.5 @ 25°C
Storage Temperature:
-20°C
Notes
General notes:- In our tests, this ligase can be used interchangeably with T4 Rnl2tr (NEB #M0242), but may require increased incubation time. Ligation activity is stimulated by adding PEG (See FAQ).
FAQs
- The molecular weight of T4 Rnl2tr K227Q is 71.6 kDa. Is it a fusion?
- Can T4 Rnl2tr K227Q be used in other NEBuffers?
- Are there differences in ligation efficiency for T4 Rnl2tr and T4 Rnl2tr K227Q?
- What can T4 Rnl2tr K227Q ligate?
- Does PEG stimulate ligation efficiency?
- How can I increase ligation efficiency?
Protocols
Protocols for T4 RNA Ligase 2, truncated K227Q
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Exonuclease Activity:
Incubation of a 50 μl reaction containing 200 units of T4 RNA Ligase 2, truncated K227Q with 1 μg of a mixture of single and double-stranded[3H] E. coli DNA (105 cpm/μg) for 4 hours at 37ºCreleased < 0.1% of the total radioactivity.
Endonuclease Activity:
Incubation of a 50 μl reaction containing 200 units of T4 RNA Ligase 2, truncated K227Q with 1 μg of ΦX174 RF I DNA for 4 hours at 37ºC resultedin < 10% conversion to RFII as determined by agarose gel electrophoresis.
Phosphatase Activity:
Incubation of 200 units of enzyme with 2.5 µmol p-nitrophenyl phosphate (PNPP) in 50 µl Reaction Buffer for 3 hours at 37°C released less than 0.05 µmol inorganic phosphate.
RNase Assay:
A 10 µl reaction in T4 Rnl2tr K227Q Reaction Buffer containing 40 ng of labeled RNA and 200 units of T4 Rnl2tr K227Q is incubated at 25°C. After incubation for 16 hours, >90% of the substrate RNA remains intact as determined by polyacrylamide electrophoresis.
References
- Yin, S., Ho, C.K. and Shuman S. (2003) J. Biol. Chem, 278, 17601-17608.
- Hafner, M., et al. (2008) Methods, 44, 3-12.
Companion Products
Low Range ssRNA Ladder
microRNA Marker
Murine RNase Inhibitor
Universal miRNA Cloning Linker
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