PBCV-1 DNA Ligase (C007B-562464)

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 20μl reaction at 25℃ in 15 minutes.This product is for R&D only. Not for drug, household, or other uses.Application：Detection of small RNAs based on probe labeling method; ligation of ssDNA splinted by complementary RNA; ligation of DNA probes to specific RNAs; detection of SNP or alternative splicing; RASL-seq analysis.Increasing the reaction temperature can improve the activity and ligation specificity of PBCV-1 DNA Ligase. The ligation efficiency of small RNAs such as microRNA can be improved by increasing the reaction temperature to 37℃.Source：PBCV-1 DNA Ligase expressed in E coli.The ligation efficiency of PBCV-1 DNA Ligase decreases with the length of the "splint" RNA (50bp-20bp). When the length of the RNA "splint" is less than or equal to 10nt, the ligation efficiency is basically zero.Enzyme storage buffer：10mM Tris-HCl (pH7.4, 25℃), 300mM NaCl, 1mM DTT, 0.1mM EDTA, 50% (v/v) glycerol.Inactivation or inhibition：Heat-inactivation at 65℃ for 20 min.Instructions for Use：1. Preparation of DNA/RNA hybridsMix two ssDNA fragments to be ligated and one complementary ssRNA in equimolar. The final concentration is recommended to be 20µM, which can be adjusted in the range of 10-50µM. Incubate at 90℃ for 1min, then anneal by gradient cooling to 25℃ to form the DNA/RNA hybrids. We recommend using 's Annealing Buffer for RNA oligos (5X) for annealing. If the DNA/RNA hybrid is not used immediately, store at -80℃.2. Set up the reaction on ice as follows.ReagentVolumeFinal ConcentrationDEPC-treated Water15µl-10X Reaction buffer2µl1XNicked DNA/RNA Substrate (1µM)2µl0.1µMPBCV-1 DNA Ligase (25U/µl)1µl1.25U/µlTotal Volume20µl-Note: Since RNA manipulation is involved, it is necessary to strictly follow the RNA manipulation specifications to avoid RNase contamination. The relevant reagents and consumables need to be RNase-free or treated with DEPC to remove RNase.3. Ligation: Incubate at 25℃ for 15-60min.4. Termination: Incubate at 65℃ for 20min; or add EDTA to inactivate the enzyme.5. Place the reaction products on ice for immediate use or store at -20℃ for future use.

Specification: Free of DNA endonuclease and exonuclease, RNase, and phosphatase.

UPC:

12352204

Condition:

New

Weight:

0.07 Ounces

HazmatClass:

No

Quantity:

1

Unit of Measurement:

EA

WeightUOM:

LB

MPN:

P748845-1250U

Product Size:

1250U