Manual

Product Number: LG09

Shipping and Storage

Stored at -20℃, valid for two years.

Component

Description

T4 RNA Ligase 2, truncated KQ is a double-point mutant (R55K, K227Q) of truncated T4 RNA Ligase 2. While maintaining ligation efficiency, it can greatly reduce non-specific ligation of RNA (RNA concatemerization or self-circularization). This enzyme specifically ligates 5′-preadenylated DNA or RNA to the 3′-OH end of RNA, and is highly suitable for ligating adapters to small RNAs for cDNA library construction. Ligation with this enzyme does not require ATP, but requires 5′-preadenylated adapters.

Unit Definition

200 units (U) is defined as the amount of enzyme required to ligate 80% of 31-mer RNA to the end of preadenylated 17-mer DNA within 1 hour at 25°C.

Inactivation Condition

65°C for 20 min.

Quality Control

1. Protein Purity: Protein purity is no less than 95% as determined by SDS-PAGE gel electrophoresis.
2. Endonuclease Activity: After incubating 200 U of T4 RNA Ligase 2, truncated KQ with 200ng of supercoiled plasmid DNA in a 20μL T4 Rnl2 KQ Buffer reaction system at 37°C for 4 hours, less than 10% of the plasmid DNA is converted to nicked or linear form as detected by agarose gel electrophoresis.
3. DNase Activity: After incubating 200U of T4 RNA Ligase 2, truncated KQ with 15ng of double-stranded DNA fragment in a 20μL T4 Rnl2 KQ Buffer reaction system at 37°C for 16 hours, no change is observed in the double-stranded DNA fragment as detected by agarose gel electrophoresis.
4. RNase Activity: After incubating 200U of T4 RNA Ligase 2, truncated KQ with 500ng of RNA in a 10μL T4 Rnl2 KQ Buffer reaction system at 37°C for 1 hour, no less than 90% of the RNA remains intact as detected by agarose gel electrophoresis.
5. Phosphatase Activity: Add 200U of this enzyme and 2.5mM p-Nitrophenyl Phosphate (pNPP) into a 200μL reaction system, incubate at 37°C for 4 hours; alkaline phosphatase activity is <0.0001 U.
6. Host DNA Residue: Determined by the quantitative PCR method (the third method) in General Rule 3407, Volume IV of the Chinese Pharmacopoeia 2020 Edition; the residual amount of E. coli host cell DNA in this product is <10 copies/200U.