T4 DNA polymerase assay

Summary

T4 DNA polymerase has a human template-dependent polymerase activity, as well as exonuclease activity for the 3' to 5' ends of single- and double-stranded DNA, and lacks exonuclease activity for the 5' to 3' ends.

Operation method

T4 DNA polymerase

Materials and Instruments

DNA
Trish-Cl dNTP MgCl2 BSA DTT
Water Bath

Move

I. 50 μl reaction volume:
(1) 50 mmol/l Tris-Cl, pH 8.0

(2) 5 mmol/l MgCl2

(3) 5 mmol/l DTT

(4) 100 μmol/l 4dNTP mixture

(5) 50 μg/ml BSA

(6) 0.1 U T4DNA polymerase

(7) 2 μg DNA

(8) The reaction was warmed at 11°C for 20 min and terminated by adding 2 μl of 0.5 mol/l EDTA or heating at 75°C for 10 min.

(9) Reactor Hoven, concentration of the four dNTp, and reaction temperature can be varied according to the specific application.II. Effects of dNTp concentration1. High concentrations of dNTP increase the ratio of polymerase activity to exonuclease activity in the usual experiments.

2. In labeling experiments, the concentration of labeled dNTP is reduced to 1 to 2 μmol/l, but the labeled dNTP should not be lower than 1 μmol/l concentration, otherwise it will lead to the degradation of its exonuclease activity pairs when dNTP is depleted.III. Degree effectsWhen labeling the 3' end with T4 DNA polymerase, the reaction temperature is kept at 11°C. At higher temperatures, its exonuclease activity tends to degrade the DNA template.

Common Problems

I. Compatibility of buffer systems

Many restriction endonucleases are able to perform cleavage reactions in the buffer system of T4 DNA polymerase, while, at the same time, T4 DNA polymerase can be used directly.
II. Applications

1. Radiolabel the 3' end of a DNA fragment containing the 5' projecting end and the appropriate [α-32P] dNTP at a concentration of 1~2 μmol/l and incubate at 11℃ for 20 min.

2. Selective and non-selective labeling linearization of the 3'-song end of the double-stranded DNA molecule, the so-called "substitution synthesis". When a double-stranded DNA fragment is incubated with T4 polymerase in the presence of dNTP, the exonuclease activity at the 3' end of the fragment results in the degradation of the DNA from the 3' end, which is activated by the addition of four types of dNTP to extend the 3' end of the DNA preservative plate. The shortest labeling effect can be achieved by adding the 4 dNTP mixture when the exonuclease activity of T4 DNA polymerase has degraded 30~40% of the outer template.

3. Change the sticky end of double-stranded DNA into a flat end, which is convenient for flat-end cloning. In the presence of a high concentration of 4dNTP, the enzymatic degradation terminates in the double-stranded region. Similarly, if there is a 5' bulge at the end, the enzyme will extend the concave 3' end until it is flush with the 5' end. In practice, DNA was incubated with T4 DNA polymerase and concentrations of 100 μmol/l dNTP each for 20 min.

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