Synthesis of fixed-length single-stranded DNA probes from M13 phage templates
The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.
Operation method
Synthesis of fixed-length single-stranded DNA probes from M13 phage templates
Materials and Instruments
E. coli DNA polymerase I Klenow fragment Restriction endonuclease Template DNA Oligonucleotide primers Move I. Materials For more product details, please visit Aladdin Scientific website.
Ammonium acetate DTT EDTA Ethanol NaCl Phenol Chloroform TBE Electrophoresis buffer Tris-Cl Klenow base buffer dNTP solution
Denaturing polyacrylamide gel or basic agarose gel Adhesive patch or phosphorescent adhesive patch Sephadex G-50 centrifuge column Water bath or heating block
1. Buffers and solutions
Ammonium acetate (10 mol/L)
DTT (1 mol/L)
EDTA ( 0.5 mol/L, pH 8.0)
Ethanol
NaCl ( 5 mol/L)
Phenol: chloroform (1:1, V/V)
5X TBE electrophoresis buffer
Tris-Cl (1 mol/L, pH 7.6)
2. Enzyme and buffer
10X Klenow basal buffer (500 mmol/L Tris-Cl ( pH 7.5), 100 mmoI/L MgSO4, filtered to remove bacteria, and stored in small portions at -20°C).
E. coli DNA Polymerase I Klenow Fragment
Restriction endonuclease suitable for the preparation of the desired probes
3. gels
Denaturing polyacrylamide gels or basic agarose gels
4. Nucleic acids and oligonucleotides
dNTP solution containing unlabeled dCTP, dGTP, and dTTP at a concentration of 20 mmol/L each
dATP (40 μmol/L and 20 mmol/L)
Oligonucleotide primers (M13 phage universal primers or customized oligonucleotides)
Template DNA
5. Radiocomplexes
[ α-32P ] dNTP ( 10 mCi/ml, specific activity 800-3000 Ci/mmol)
6. Specialized equipment
Radioactive ink labeled adhesive patches or phosphorescent adhesive patches (e.g. Glogos, Stratagene)
Beaker (250 ml) with water at 85°C and microcentrifuge tube holder
Sephadex G-50 centrifuge column equilibrated with TE (pH 7.6)
Preheated water baths or heating blocks at 68 °C and 85 °C
Preheat the water bath to the appropriate temperature for restriction endonuclease digestion
ii. Methods
1. Mix in a 0.5 ml microcentrifuge tube:
Single-stranded template (M13 phage or phage DNA) ( approx. 0.5 pmol) 1 μg
Oligonucleotide primer 5 pmol
10X Klenow base buffer 3 μl
Water Add to 20 μl
2. Heat the reaction mixture at 85°C for 5 min, then slowly cool to 37°C.
3. Add to the test tube containing the primer and template for complexation:
0.1 mol/L DTT 2 μl
10 mCi/ml [ α-32P ] dATP
(specific activity 3000 Ci/mmol) 5 μl
40 μmol/L dATP 1 μl
20 mmol/L dTTP, dcTP and dGTP solution 1 μl
Tap the outside of the tube to mix the components. Centrifuge in a microcentrifuge at maximum speed for 1~2s to settle all liquid to the bottom of the tube.
4. Transfer 0.5 μl of the mixture to a microcentrifuge tube containing 15 μl of 20 mmol/L EDTA (pH 8.0) and place the tube on ice.
5. Add 1 μl ( 5 units) of Klenow Fragment to the remaining reaction mixture. Tap the walls of the tube to mix the reaction components. Incubate the reaction for 30 min at room temperature.
6. Transfer 0.5 μl of the reaction to a new microcentrifuge tube containing 20 μl of 0.5 mol/L EDTA (pH 8.0). Place the tube on ice.
7. Add 1 μl of 20 mmol/L unlabeled dATP to the remaining reaction solution and flick the outside of the tube to mix the reaction. Centrifuge at maximum speed for 1-2 s to settle all liquid to the bottom of the tube. Continue to incubate for 20 min at room temperature.
8. During the 20 min incubation (Step 7), use the samples saved from Steps 4 and 6 to determine the ratio of radioactivity of molecules transferred to the 10% trichloroacetic acid (TCA) precipitates or attached to the DE-81 membrane.
9. Inactivate the Klenow fragments by heating the reactants at 68 °C for 10 min.
10. Adjust the concentration of NaCl in the reaction to match the optimal digestion conditions for the restriction endonuclease of choice. Remember to use 50 mmol/L NaCl in the primer extension reaction.
11. Add 20 units of the desired restriction endonuclease and incubate for 1 h at the appropriate temperature.
12. Purify DNA by standard phenol/chloroform extraction and remove unincorporated dNTP by centrifugal column chromatography or by graded precipitation with 2.5 mol/L ammonium acetate and ethanol. add 0.5 mol/L EDTA (pH 8.0) to a final concentration of 10 mmol/L.
13. Depending on the fragment size, use denaturing polyacrylamide gel or basic agarose gel electrophoresis to separate the radiolabeled DNA.
14. After electrophoresis, the gel is prepared for radioactive autoradiography. Expose the gel to X-ray film for 5~10 min.
15. After the negative has been developed, the developing film is aligned with the gel using Glogos or radioactive ink spots. Place the film on the gel and mark the position of the primer-extended radioactive DNA fragment on the back of the gel plate. Remove the film and cut off the gel block containing the desired DNA fragments.
16. If a polyacrylamide gel is used to separate the DNA fragments from the template, proceed to step 17. If an alkaline agarose gel is used, the gel should be neutralized by gently shaking for 45 min in 0.5 mol/L Tris-Cl (pH 7.6), followed by shaking for 45 min in 0.5X TBE, prior to elution of the radiolabeled DNA.
17. Extract the DNA from the gel by electroelution or by squeezing the polyacrylamide gel block and soaking it in an appropriate buffer for DNA extraction. 
