The experiment was designed to synthesize two complementary DNA Oligos. Small DNA fragments with 5 'sticky ends were synthesized by annealing, and the sticky ends were completed by T4 DNA Polymerase to form flat ends. 20% Native PAGE glue (which can distinguish the size of 0.1~100bp) was used for electrophoresis, and the enzyme activity was verified. As can be seen from the result, the dsDNA fragment at the sticky end is mended by T4 DNA Polymerase, which has a larger molecular weight, and the electrophoretic band position is above the DNA fragment with the sticky end.
Lane 1 Negative Control
Lane 2 UA070070-T4 DNA Polymerase 10ng
Lane 3 UA070070-T4 DNA Polymerase 20ng
Lane 4 UA070070-T4 DNA Polymerase 40ng
Lane 5 UA070070-T4 DNA Polymerase 80ng
Lane 6 UA070070-T4 DNA Polymerase 0.1ug
Lane 7 UA070070-T4 DNA Polymerase 0.2ug
Lane 8 UA070070-T4 DNA Polymerase 0.4ug
Lane 9 UA070070-T4 DNA Polymerase 0.8ug
T4 DNA Polymerase
T4 DNA Polymerase
Product Details
Product Details
Product Specification
| Synonyms | DNA-directed DNA polymerase |
| Expression System | E.coli |
| Molecular Weight | 105kDa (Reducing) |
| Purity | ＞95% by SDS-PAGE and HPLC |
| Conjugation | Unconjugated |
| Tag | His Tag |
| Physical Appearance | Liquid |
| Storage Buffer | 100 mM K3PO4, 1 mM DTT, 50% Glycerol, pH 6.5 @ 25°C |
| Stability & Storage | Store at -25 ~ -15℃ for 2 years |
| Reference | 1. Capson TL, Peliska JA, Kaboord BF, Frey MW, Lively C, Dahlberg M, Benkovic SJ. Kinetic characterization of the polymerase and exonuclease activities of the gene 43 protein of bacteriophage T4. Biochemistry. 1992 Nov 17;31(45):10984-94. |
Background
T4 DNA polymerase catalyzes the synthesis of DNA in the 5´→3´ direction and requires the presence of template and primer. This enzyme possesses 3´→5´ exonuclease activity, which is much higher than that found in DNA polymerase I (E. coli). Unlike E. coli DNA polymerase I, T4 DNA polymerase lacks 5’ →3’ exonuclease activity. T4 DNA polymerase can be used to Removal of 3’ overhangs or fill-in of 5’ overhangs to form blunt ends.
Components
3 U/μl T4 DNA Polymerase, 100 mM K3PO4, 1 mM DTT, 50% Glycerol, pH 6.5 @ 25°C
10* Reaction Buffer: 500 mM NaCl, 100 mM Tris-HCl, 100 mM MgCl2, 1000 µg/ml Recombinant Albumin, (pH 7.9 @ 25°C)
Protocol
Incubate the following reaction at 12°C for 15 minutes
Component |
Final Concentration |
10X Reaction Buffer |
1X |
dNTP Mix (10 mM) |
100 mM each |
T4 DNA Polymerase (3,000 U/ml) |
1000 U/ml |
DNA |
10 ug |
Nuclease-free Water |
to 10 µl |
Total Reaction Volume |
10 µl |
Stop reaction by adding EDTA to a final concentration of 10 mM and heating to 75°C for 20 minutes
Guidelines
Elevated temperatures, excessive amounts of enzyme, failure to supplement with dNTPs or long reaction times will result in recessed ends due to the 3´ → 5´ exonuclease activity of the enzyme.
Unit Definition
Picture
Picture
Bioactivity
SDS-PAGE
2μg (R: reducing condition, N: non-reducing condition).
RP-HPLC
