During synthesis, low concentrations of radiolabeled dNTP limit the length of probes to 200-300 nucleotides. However, newly synthesized DNA can be made 100-1000 nucleotides long by varying the amount of template input and the concentration of dNTP, and probes in this length range are suitable for most hybridization experiments. This experiment is based on the "Guide to Molecular Cloning, Third Edition", translated by Huang Peitang et al.
Operation method
Synthesis of non-fixed-length single-stranded DNA probes from M13 phage templates
Principle
During synthesis, low concentrations of radiolabeled dNTP limit the length of probes to 200-300 nucleotides. However, newly synthesized DNA can be made 100-1000 nucleotides long by varying the amount of template input and the concentration of dNTP, and probes in this length range are suitable for most hybridization experiments.
Materials and Instruments
E. coli DNA polymerase I Klenow fragment Oligonucleotide primer Template DNA Move I. Materials For more product details, please visit Aladdin Scientific website.
Elution buffer Formamide staining buffer NaOH probe synthesis buffer
Polyacrylamide gel Air incubator or water bath shaker Boiling water bath Disposable inoculation sticks Heating plates Plastic snap-cap tubes
1. Buffers and solutions
Elution buffer (1X SSPE with 0.5% SDS)
Formamide staining buffer
MgCl2 ( 1 mol/L)
NaOH (10 mol/L)
3X probe synthesis buffer (dATP, dTTP, and dGTP (62 pmol/L each), 0.5 mg/ml bovine serum albumin (fraction V, Sigma), 625 μmol/L DTT, 32 mmol/L Tris-Cl (pH 7.5), 6.5 mmol/L MgSO4 )
2. Enzyme and buffer
E. coli DNA polymerase I Klenow fragment
3. gel
Polyacrylamide gel with 7 mol/L urea (5 %)
4. nucleic acids and oligonucleotides
Oligonucleotide primers (M13 phage universal primers or customized oligonucleotides)
Template DNA
5. Radiocomplexes
[ α-32P ] dCTP ( 10 mCi/ml, 800~3000 Ci/mmol)
6. Specialized equipment
Air incubator or water-bath shaker preheated to 50℃.
Boiling water bath
Disposable inoculation sticks
Multi-well (fritted) column (Quik-sep column, Isolab)
Heating plate preheated to 57°C
Plastic snap-cap tubes (12 X 17 mm)
Methods
1. Mix in a 0.5 ml microcentrifuge tube:
Single-stranded template (M13 phage or phage DNA)
( ~0.15 pmol, ~0.1 μg/μl) 0.3 μg
Oligonucleotide primer 1 μl
25 mmol/L MgCl 1 μl
If possible, set up a positive control reaction in each experiment containing either a control DNA template or an oligonucleotide that has been experimentally confirmed to react well.
2. Cover the tube tightly and incubate the reaction on a heating plate at 57℃ for 5~10 min.
3. Transfer the microtube from the heating plate to ice and add the following mixture:
3X Probe Synthesis Buffer 4 μl
10 mCi/ml [ α-32P ] dCTP
( specific activity 3000 Ci/mmol) 3 μl
Klenow fragment (approx. 2.5 units) 0.5 μl
Tap the wall of the tube to mix the components, centrifuge at maximum speed for 1~2s to allow all liquid to settle to the bottom of the tube, and incubate the reaction at 37°C for 40 min.
4. A 5% polyacrylamide gel containing 7 mol/L urea (prepared with 1X TBE buffer) can be prepared at the same time as the primer reaction. The thickness of the gel should be 1.5 mm, the length should be at least 15 cm, and the width of the spiking wells should be 2.5 cm. Electrophoresis at 20~25 V/cm for 15 min should be performed to remove amine persulfate from the spiking wells.
5. Add 25 μl of formamide dye buffer and heat the reaction in a boiling water bath for 3~5 min to terminate the primer extension reaction. Transfer the microtube to ice and add 1 μl of 1 mol/L NaOH.
6. Aspirate 1X TBE buffer with a syringe, wash out the urea and loose polyacrylamide gel fragments in the spiking wells, and then immediately add the DNA samples into the spiking wells. Electrophoresis for 30 min at 20~25 V/cm to separate the template DNA from the radiolabeled probe.
7. After 30 min of electrophoresis, the gel is separated and removed, wrapped in Saran Wrap, and the radiolabeled DNA is localized by radioactive autoradiography, and the gel is exposed to X-ray film for 1 min.
8. Use a clean razor blade to cut the radioactive zone from the gel and place it in the bottom of a 12 x 17 mm plastic tube with a snap-cap, mash it with a disposable inoculation stick, add 1-2 ml of elution buffer, and shake it at 50°C for >3 h. After 30 min of electrophoresis, the gel is removed and wrapped in Saran Wrap packaging film to localize the radiolabeled DNA by radioautography.
9. Pipette the eluate into a multiwell (fritted) column, place the column in a plastic snap-cap tube, and centrifuge it in a tabletop centrifuge for 1~2 min. 1 μl of radioprobe was detected for radioactivity with a liquid scintillation counter. 