Labeling oligonucleotide probes with T4 phage polynucleotide kinase
The synthetic oligonucleotide lacks a phosphate group at its 5 end during synthesis, so it is very easy to use T4 phage polynucleotide reaction. This probe can reach γ = 32P from [γ = 32P] ATP itself with high radiation Specific activity. The reaction described below is designed for labeling 10pmol oligonucleotides with high specific activity. By expanding or reducing the scale of this reaction, different amounts of oligonucleotides can be successfully labeled, while the concentration of each component can be kept constant. cat toys interactive,cat toys wand,cat toys balls,cat teething toys Ningbo XISXI E-commerce Co., Ltd , https://www.petspetskitty.com
1) Prepare the following reaction mixture:
Oligonucleotide (10 pmol/μl) 1.0μl
10xT4 phage polynucleotide enzyme buffer 2.0μl
[γ = 32P] ATP (specific activity 5000Ci / mmol; 10mCi / ml aqueous solution) (10pmol) 5.0μl water 11.4μl
10xT4 phage polynucleotide kinase buffer
0.5mol / L Tris.Cl (pH7.6)
0.1mol / L MgCl2
50mol / L dithiothreitol (DTT)
1mol / L iminochloride
1mol / L EDTA (pH 8.0) was mixed well. Add 0.5μl of the above reaction mixture to a reaction tube containing 10μl of 10mol / L Tris.Cl (pH 8.0) and set aside for use in step 3). The concentration of [γ = 32P] ATP and oligonucleotide contained in this reaction is equal, and only 50% of the radioactive label is transferred to the oligonucleotide. If the concentration of oligodic acid in the reaction is increased to 10 times, the efficiency of transfer can be improved. As a result, nearly 90% of the radioactive label is transferred to the oligonucleotide. However, the specific activity of the oligonucleotide will be reduced to 1/5 of the original. If high specific activity labeling is performed on a segment of oligo acids, you can:
a. Increase the concentration of [γ = 32P] ATP in the reaction by 3 times (that is, use 15 μl of radioactive label in the reaction mixture and reduce the volume of water to 1.4 μl.
b. Reduce the oligonucleotide concentration to 3 pmol. In these cases, only about 10% of the radiolabel is transferred, but in fact each oligonucleotide molecule is labeled.
2) Add 8 units (about 1 μl) of T4 phage polynucleotide kinase to the reaction mixture. Mix well and incubate at 37 ° C for 45 minutes. Another 0.5 μl of this reaction solution was added to another reaction tube containing 10 10 mmol / LTris.Cl (pH 8.0), and set aside for use in step 3). The remaining reaction solution was heated at 68 ° C for 10 minutes to inactivate T4 phage polynucleotide kinase.
3) According to the following method (Stawinski et al., 1977; Narang et al., 1980), determine the efficiency of 32 P transfer to oligonucleotide and estimate its specific activity:
a. Cut a piece of cellulose paper impregnated with polyethyleneimine (Polygram CEL 300EI: Brinkmann) about 15 cm long and about 5 cm wide. Use a soft lead pencil to draw a thin straight line across the strip at a distance of 2.5 cm from one end. Make two marks on the line. The distance between the two marks is about 1 in (2.54 cm).
b. Click 0.5μl of the liquids set aside in steps 1) and 2) at the two marks respectively, and place the paper strip vertically in a chromatography dish so that the radioactive sample is at the lower edge of the paper strip. The bottom of the paper strip should extend into a dish containing a development buffer (0.5 mol / L ammonium bicarbonate) with a height of about 0.5 cm. Cover with chromatography captivity. Expand the chromatography until the solvent front migrates about 4-5 in (10.16-12.7 cm).
c. Wrap the polyethyleneimine-cellulose paper strips in Saran packaging film for autoradiography, or cut the paper strips horizontally into narrow (0.25cm) strips, and measure each narrow strip in a liquid scintillation counter Radioactivity. Oligonucleotides remain at the starting point, while ATP and inorganic phosphate will move in the same direction as the solvent. Inorganic phosphoric acid migrates slightly slower than the solvent front, and the position of ATP is almost equidistant from the starting point and inorganic phosphoric acid. In this way, if phosphate is transferred from γ-32P] ATP to the oligonucleotide, it will cause radioactivity at the origin. By measuring the radioactivity value at the starting point and the entire paper strip, the percentage of radioactive label transferred from γ-32P] ATP to the oligonucleotide can be calculated. The specific activity of the probe can be calculated based on the moles of Hu nucleotide and γ-32P] ATP in the reaction. Under the above reaction conditions, nearly 50% of the radioactivity should be transferred to the oligonucleotide, and the specific activity was close to 2500 Ci / mmol. The radiolabel transferred to the oligonucleotide can also be determined by the DE-81 filter absorption method. The oligonucleotide is firmly bound to the positively charged filter, while the unincorporated radioactive label can be removed by repeated washing with sodium phosphate solution.
4) If the specific activity of the probe meets the requirements, continue the purification of the radiolabeled synthetic oligonucleotide stack. If the specific activity is too low, add another 8 units of enzyme, continue to incubate at 37 ℃ for 30 minutes (that is, 75 minutes in total), and heat at 68 ℃ for 10 minutes to inactivate the enzyme, and analyze the product of the reaction again.