5' DNA/RNA Adenylase

Item Number

D743509

• Specifications & Purity: Free of DNA endonuclease and exonuclease, free of RNase.
• Stability And Storage: Store at -20℃, valid for at least 2 years.
• Storage Temp: Store at -20°C
• Shipped In: Ice chest + Ice pads

Product Description

Aladdin's 5' DNA/RNA Adenylase catalyzes the conversion of 5' phosphorylated single-stranded DNA or single-stranded RNA (pDNA or pRNA) to 5' adenylated DNA/RNA (AppDNA/RNA). Regardless of whether the 3' end of DNA/RNA is blocked by amination or not, 5' adenosylation can be efficiently produced. In the absence of ATP, this product can catalyze the formation of circular DNA from 5' phosphorylated single-stranded DNA (pDNA).The working principle of 5' DNA/RNA Adenylase is as follows

Application：

Preparation of 5' adenylated ssDNA linkers which are eventually added to 3'-OH group of miRNA, other RNA, or ssDNA for cloning, NGS library preparation, or PCR etc.

Source：

Adenosine acylase of thermophilic bacteria expressed and purified from E. coli.

Inactivation or inhibition：

5' DNA/RNA Adenylase can be inactivated by heating at 85℃ for 5min. When using a reaction volume of 20μl, R7019S and R0719M are sufficient for 40 and 200 reaction.

Precautions：

Phosphorylation of the 5' end of ssDNA or ssRNA is required, while blocking the 3' end by amination or other modifications is unnecessary. when the 3' end is not blocked, increase the concentration of ATP to 0.5 mM in the reaction to avoid the cyclization or tandem reaction of the substrate.5' DNA/RNA adenylase cannot ligate dsDNA nicks.The optimal working temperature for 5' DNA/RNA adenylase is 65℃. Deadenylation occurs at 25℃, thus incubation at 85℃ for 5 min is recommended to inactivate the 5' DNA/RNA adenylase after the adenylation is complete. If the 5' DNA/RNA adenylase is not fully inactivated, subsequent exposure to room temperature conditions will result in a decrease in adenylation.Keep the 5' DNA/RNA adenylase on ice during the operation, and store at -20℃ immediately after use.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation.

Instructions for Use：

1. Thaw the reagents required for the adenylation reaction. Keep 5' DNA/RNA adenylase on ice. 2. Set up the following reaction in a sterile microfuge tube on ice.Reagent Volume Final Concentration Nuclease-free Water14µl -ssDNA or ssRNA (100µM) 1µl 5µM 10X Adenylase Reaction Buffer 2µl 1X ATP (1mM) 2µl 0.1mM 5' DNA/RNA adenylase (75U/µl) 1µl 3.75U/µl Total Volume 20µl -Note: a. When multiple reactions are required, prepare a master mix including all reagents except the ssDNA or ssRNA substrate and then dispense to different nuclease-free tubes. Finally, add the ssDNA or ssRNA substrate into each tube. b. If RNA manipulation is involved, it is necessary to use RNase-free reagents and consumables to avoid any RNase contaminations. If single-stranded RNA is involved, we recommend adding an appropriate amount of RNase Inhibitor (, R0102) in the reaction.3. Incubate at 65°C for 1 hour. The reaction time can be extended appropriately to make the adenylation more thoroughly.4. Terminate the reaction by heating at 85℃ for 5 min.5. Examination of adenylated products by electrophoresis: Denature the reaction product by incubating at 95℃ for 5min followed by an immediate cooling on ice. Use a denaturing polyacrylamide gel (15%) containing 7M urea for electrophoresis. The molecular weight will become a little larger after adenylations (Figure 1).6. Concentration and subsequent applications: The reaction product can be concentrated by conventional ethanol precipitation method and subsequently used for ligation with 3' hydroxyl group of small RNA under the catalysis of T4 RNA Ligase2, truncated (200U/µl) or other appropriate ligases.