Deoxyribonuclease I (DNase I), GMP-grade HYC116
DNase I is an endonuclease that can digest single-stranded and double-stranded DNA to produce single deoxynucleotides or single-stranded or double-stranded oligodeoxynucleotides.It can hydrolyze the phosphodiester bond to produce monodeoxynucleotides and oligodeoxynucleotides containing 5'-phosphate groups and 3'-OH groups. The average digestion product is the smallest polytetranucleotide. DNase I can catalyze many forms of DNA, such as single-stranded DNA,double-stranded DNA, and even chromatin(its cutting rate is affected by histones).The optimum pH range is 7-8. The activity of DNase I depends on Ca²+ and can be activated by divalent metal ions,such as Co2+, Mn²+, Zn²+, etc. 5mM Ca²+ can protect the enzyme from being hydrolyzed. In the presence of Mg²+, the enzyme can recognize and cut any site on any strand of DNA randomly;and in the presence of Mn²+, it can recognize two strands of DNA at the same time and cut at almost the same site to form blunt ends, or sticky ends with 1-2 nucleotides protruding. This product is produced in accordance with GMP process requirements, and the product is provided in liquid form.
Components
|
Components |
500 U |
2000 U |
10000 U |
|
Deoxyribonuclease I (DNase I), GMP-grade |
250μL |
1mL |
5mL |
Storage
Store at-15°C ~-25℃ for one year.
Unit Definition
The amount of enzyme required to completely degrade 1μg of plasmid DNA within 10 min at37℃. (The reaction buffer is: 10 mM Tris-HCl pH7.6, 2.5mM MgCl2,0.5mM CaCl₂, 1μg plasmid DNA)
Specification
|
Source |
Recombinant E.coli with DNase I gene |
|
Optimum temperature |
37℃ |
|
Storage Buffer |
10 mM Tris-HCl pH7.6, 2mM CaCl2, 50%(v/v) Glycerol |
Instructions
1. Plasmid template digestion
1）Reaction system:
Use the RNase-free centrifuge tube and pipette tip to prepare the following reaction system:
|
Components |
Volume |
|
10×DNase I Buffer* |
1μL |
|
DNase I |
1μL |
|
RNA |
X |
|
RNase-free ddH₂O |
Up to 10μL |
*10×DNase I Buffer: 10 mM Tris-HCl, 2.5mM MgCl2, 0.5mM CaCl2, pH7.6 at 25℃.
2) Reaction conditions:
37℃, 15-30min later, add a final concentration of 2.5mM EDTA solution and mix well at 65℃ for 10min. The processed template can be used in subsequent reactions such as capping reaction.
2. DNase I inactivationor inhibition
After adding EDTA to a final concentration of 2.5mM, heating at 65℃ for 10 min can inactivate DNase I. Phenol and chloroform extraction can also inactivate DNase I. The following conditions all have significant inhibitory effect on DNase I: Metal ion chelating agents, zinc ions with a concentration of millimoles/liter, 0.1%SDS, DTT, mercaptoethanol and other reducing agents, the salt concentrations above 50-100 mM.
Applications
Preparation of DNA-free RNA; Remove the template DNA after in vitro transcription; Prepare DNA-free RNA before RT-PCR and RT-qPCR reactions; Combine with DNA polymerase I to perform DNA labeling through nick translocation; DNA fragmentation library construction.
Notes
1. Enzymes should be stored in an ice box or on an ice bath when used,and should be stored at -20℃ immediately after use.
2. For your safety and health,please wear personal protective equipment(PPE),such as laboratory coats and disposable gloves, when operating with this product.