商品编号


EG-1063



品牌


Excellgen



公司


Excellgen, Inc.



公司分类


Ligases




1,000 Units

商品信息



Description


Chlorella virus PBCV-1 DNA ligase (Chlorella virus DNA ligase, also known as SplintR? Ligase sold by
NEB
), is an ATP-dependent DNA ligase. PBCV-1 DNA ligase efficiently catalyzes the ligation of adjacent, single-stranded DNA splinted by a complementary RNA strand. This unusual activity can be used to characterize miRNAs, RNAs and SNPs, e.g. sensitive (sub-nanomolar range) detection of specific RNA by ligation. In addition, PBCV-1 DNA ligase may be used for enrichment of RNA for next-generation sequencing.

PBCV-1 DNA ligase enzyme is active over a broad range of ATP concentrations (10 ?M – 1 mM) and pH (6.5–9). Optimal activity is observed using Mg2+ > 5 mM, and a pH between 7.5 and 8.0. The activity is enhanced at elevated temperatures (up to 37°C), and by supplementation with 5 mM Mn2+. The reaction is inhibited by salt concentrations when concentration is > 100 mM.

The enzyme tolerates all base pair combinations at the ligation junction, but is partially inhibited by dC/G and dG/C base pairs at the donor (phosphorylated) side ligation junction, particularly when the +2 base was also a C/G base pair.




Applications



Ligation of ssDNA splinted by complementary RNA sequences

Detection of RNA using ligation of DNA probes

SNP or splice variant detection

RASL-seq





Source

Purified from a recombinant
E. coli
strain carrying the PBCV-1 DNA ligase gene



Components



Chlorella virus PBCV-1 DNA ligase
,
10,000 units
in storage buffer:
10 mM Tris-HCl, 300 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 50% Glycerol, pH 7.4 @ 25°C

10 X Ligation Buffer:
250 mM Tris-HCI, 50 mM MgCl
2
, 5 mM DTT, 10 mM ATP, pH 7.6 @ 25°C





Unit Definition

One unit is defined as the amount of enzyme needed to ligate (to 50% completion) 2 picomoles of a tripartite FAM-labeled DNA:RNA hybrid substrate in a 20 μl reaction at 25°C in 15 minutes in 1X Ligation Buffer.



Quality Control


The absence of endo-, exodeoxyribonucleases, and ribonucleases confirmed by appropriate quality tests.




Storage

-20°C.