DNA polymerase I, Large (Klenow) Fragment
Cat. No.: SB-G3457-100U
Size: 100U
Description
DNA polymerase I, Large (Klenow) Fragment, also known as Klenow fragment, is a large fragment derived from Escherichia coli DNA polymerase I, which is recombinantly expressed in Escherichia coli. It retains the 5'-3' polymerase activity and 3'-5' exonuclease activity of DNA polymerase I, and lacks the 5'-3' exonuclease activity. Based on these characteristics, it is mainly used for the blunt end of the 5' or 3' protruding end of double-stranded DNA, 5' end labeling, Sanger dideoxy method for DNA sequencing, cDNA second-strand synthesis, site-directed mutagenesis, etc.
- Source : derived from Escherichia coli, recombinantly expressed by Escherichia coli.
- Definition of enzyme activity: at 37°C, the amount of enzyme required to catalyze the incorporation of 10 nmol deoxyribonucleotides into polynucleotides within 30 minutes is defined as one enzyme activity unit.
- Purity and concentration: SDS-PAGE detection purity ≥ 95%; endogenous nucleic acid residue < 1 pg/μL (qPCR detection); 2 U/μL.
- Inactivation or inhibition: Heating at 75°C for 20 min or adding an appropriate amount of EDTA can lead to enzyme inactivation. Metal ion chelating agents and inorganic pyrophosphates can inhibit enzymes.
- Enzyme Storage Buffer: 25 mM Tris-HCl, 1 mM DTT, 0.1 mM EDTA, 50% Glycerol, pH 7.4.
- 10x Reaction Buffer: 100 mM Tris-HCl, 500 mM NaCl, 100 mM MgCl2 , 10 mM DTT, pH7.9.
Storage and transportation
Transport in wet ice packs; store at -20°C, valid for 12 months.
Product Contents
| Component Number |
Component |
G3457 |
| G3457-1 |
DNA polymerase I, Large (Klenow) Fragment |
50 μL |
| G3457-2 |
10x Reaction buffer |
500 μL |
| Product Manual |
1 copy |
Steps
Fill in dsDNA 5' protruding ends:
1. Configure the reaction system according to the following table
| Component |
Volume |
| DNA sample |
0.1-4μg |
| DNA polymerase I, Large (Klenow) Fragment |
1-4U |
| dNTP Mix (2.5 mM each) |
0.4 μL |
| 10x Reaction Buffer |
2 μL |
| Nuclease Free Water |
To 20 μL |
2. After the system is prepared, mix well and incubate at 37°C for 10 min. The reaction was then terminated by incubating at 75°C for 10 min.
For the labeling reaction of the dsDNA 5' protruding end, just replace the dNTP Mix in the above reaction with the labeled dNTP Mix for the reaction.
Precautions
1. All enzymes should be stored in an ice box and stored at -20°C immediately after use.
2. For your safety and health, please wear a lab coat and disposable gloves for operation.
