T4 RNA Ligase 1

• ATP-dependent ligation of single-stranded RNA and single-stranded DNA

T4 RNA Ligase 1 catalyzes the ATP-dependent ligation of single-stranded nucleic acids (RNA or DNA).

T4 RNA Ligase 1 is supplied in 10 mM Tris-HCl, 50 mM KCl, 0.1 mM EDTA, 1.0 mM DTT and 50% glycerol; pH 7.5 at 25°C.
10X T4 RNA Ligase Buffer (cat. no. B6050) contains the following: 500 mM Tris-HCl, 100 mM MgCl₂, 10 mM ATP and 100 mM DTT; pH 7.8 @ 25°C.

Enzyme properties

• Storage temperature: –25°C to –15°C
• Molecular weight: 43,509 Daltons

T4 RNA Ligase 2 Truncated

References

1. Ho, C.K., et al. (2004) Structure 12:327.
2. Ho, C.K., and Shuman, S. (2002) Proc. Natl.Acad.Sci. USA 99:12709.
3. Nandakumar, J., et al. (2004) J. Biol. Chem. 279:31337.
4. Aravin, A., and Tusch, T. (2005) FEBS Letters 579:5830.
5. Pfeffer, S., et al. (2005) Nat. Meth. 2:269.

Source of recombinant enzyme protein

The T4 RNA 1 protein is produced by a recombinant E. coli strain carrying the T4 RNA Ligase gene from bacteriophage T4.

Unit definition

One unit is defined as the amount of enzyme required to ligate 50% of 0.4 µg of an equimolar mix of two single-stranded 23 base RNA oligonucleotides (one 5′-phosphorylated) in 20 µl 1X T4 RNA Ligase Buffer following a 30 minute incubation at 37°C.

Quality control analysis

Specific activity T4 RNA Ligase 1 was measured using a twofold serial dilution method. Dilutions of enzyme were made in 1X T4 RNA Ligase reaction buffer and added to 20 µl reactions containing 0.4 µg of an equimolar mix of two single-stranded 23 base RNA oligonucleotides (one 5′-phosphorylated) and 1X T4 RNA Ligase Buffer. Reactions were incubated 30 minutes at 37°C, stopped, and analyzed on a 15% TBE-Urea gel stained with SYBR^® Gold Nucleic Acid Gel Stain (Invitrogen S-11494).

Protein concentration is determined by OD₂₈₀ absorbance.

Physical purity is evaluated by SDS-PAGE of concentrated and diluted enzyme solutions followed by silver-stain detection. Purity is assessed by comparing the aggregate mass of contaminant bands in the concentrated sample to the mass of the band corresponding to the protein of interest in the diluted sample.

Single-stranded exonuclease is determined in a 50 µl reaction containing a radiolabeled single-stranded DNA substrate and 10 µl of enzyme solution incubated for 4 hours at 37°C.

Double-stranded exonuclease activity is determined in a 50 µl reaction containing a radiolabeled double-stranded DNA substrate and 10 µl of enzyme solution incubated for 4 hours at 37°C.

Double-stranded endonuclease activity is determined in a 50 µl reaction containing 0.5 µg of plasmid DNA and 10 µl of enzyme solution incubated for 4 hours at 37°C.

E. coli contamination is evaluated using 5 µl replicate samples of enzyme solution that are denatured and screened in a TaqMan qPCR assay for the presence of contaminating E. coli genomic DNA using oligonucleotide primers corresponding to the 16S rRNA locus.

Non-specific RNAse contamination is assessed using the RNAse Alert kit, (Integrated DNA Technologies), following the manufacturer’s guidelines.

This OEM by QIAGEN product is available for bulk purchase for the following commercial assay applications.

• Ligate single-stranded RNA and single-stranded DNA
• Label 3’ termini of RNA