Polymerase Chain Reaction, abbreviated as PCR (English full name: Polymerase Chain Reaction) (also known as: Polymerase Chain Reaction), Polymerase Chain Reaction, is an in vitro enzyme-promoted synthesis of specific DNA fragments by high-temperature denaturation, low-temperature annealing (denaturation) and temperature extension of the several steps of the reaction composed of a cycle, cyclic, so that the purpose of the DNA can be rapidly amplified, with high specificity, sensitivity, easy to operate, time-saving features. It is characterized by high specificity, high sensitivity, easy operation and time saving.
Operation method
basic program
Materials and Instruments
Probe Move 1. Prepare the following hybridization mixture(1) 2 μl 200 pmol/l probe (final concentration 4 pmol/l)(2) 50 μl deionized formamide (final concentration 50%)(3) 10 μl 20× SSC (final concentration 2×)(4) 10 μl of 100 x Denhardt solution (final concentration 10 x)(5) 10 μl 10 mg/ml sonicated salmon sperm DNA (final concentration 1 mg/ml)(6) 1 μl 10% SDS (final concentration 1%)(7) 7 μl water2. Add 10 μl of hybridization mixture to each sample well of the slide containing nucleic acids amplified by in situ PCR. Cover each well with a coverslip and place on a 95℃ heating block for 5 min. For more product details, please visit Aladdin Scientific website.
SSC SDS Developing Solution Fixing Solution Hematoxylin PBS AEC Blocking Solution NaCl Tris-Cl Dimethylformamide BCIP NBT Ethanol
Incubator Coverslips Slides Drying agents Humidifying boxes
3. Place the slide in a humidified box and incubate at 48℃ for 2~4 hours.If a 33P-labeled probe is used:
4a. Remove the coverslip and wash the slides in 2× SSC for 5 min.
5a. Dip the slide in diluted Kodak latex.
6a. air dry, then place in a closed-light slide box with desiccant for 3-10 days.
7a. In a darkroom, develop the slides according to the following procedure:(1) Sequentially immersed in Kodak developer, 3 min
(2) Water, 30 s
(3) Kodak Unifix fixative, 3 min8a. The slides were re-stained by warming for 2-3 min at room temperature in 2% Gills hematoxylin staining solution.Such as with biotin or digoxin labeled probes detected by peroxidase:
4b. Remove the coverslip and wash the slide twice in PBS for 5 min per dip.5b. Add 10 μl of 100 μg/ml streptavidin-peroxidase solution to each well of the slide, gently cover the coverslip, and incubate at 37℃ for 1 h.
6b. Remove the coverslips and wash the slides twice in PBS for 5 min each time.7b. In the dark, add 100 μl of AEC working solution to each well of the slide, incubate at 37°C for 10 min, then observe under the microscope, and if the color is not strong enough, extend the color development for another 10 min.
8b. Dip the slides in tap water and dry them.Such as with biotin or digoxin labeled probes detected by alkaline phosphatase:4c. Remove the coverslip and wash the slide twice in 2×SSC at room temperature for 15 min each time, then add 100 μl of blocking solution to the sample wells, cover the surface of each well, and lay the slide flat in a humidified box and incubate for 15 min at room temperature.
5c. For each well to be chromatographed, mix 10 μl of 40 μg/ml Streptavidin-Phosphatidylphosphatase Conjugate with 90 μl of Conjugate Dilution Buffer.6c. Touch the edge of the slide with absorbent paper, absorb the blocking solution, add 100 μl of conjugate solution diluted according to the previous step to each well, lay the slide flat in a humidified box and incubate for 15 min at room temperature.
7c. Wash the slides twice in 100 mmol/l Tris-Cl pH 7.5/150 mmol/l NaCl for 15 min each at room temperature, and then wash the slides once (5 min) in alkaline phosphatase substrate buffer at room temperature.
8c. Preheat 50 ml of alkaline phosphatase substrate buffer in a Coplin wide-mouth flask to plus 37°C, add 200 μl of 75 mg/ml NBT and 166 μl of 50 mg/ml BCIP and mix thoroughly. The slides were then incubated in this solution at 37°C until the desired degree of color development was achieved. (Usually 10 min~2 h, the slide can be intermittently removed from the solution to observe the degree of color development under a 10× objective, but do not allow the slide to dry out.) Then, the reaction was terminated by immersing the slide in deionized water, during which the water was changed several times.9. Stain the slides for 5 min at room temperature with 0.2% Gills hematoxylin (if used for peroxidase-based color development) or 1% nucleic acid solid red stain (if used for alkaline phosphatase-based color development), immerse the slides in tap water, and change the water several times.
10. The slides were dehydrated by incubation in 50%, 70%, 90% and 100% ethanol for 1 min at room temperature and then air-dried.
11. Add 1 drop of fixative per well, press the coverslip and observe the results immediately (be careful not to move the coverslip), or leave at room temperature overnight to allow the fixative to dry.
Figure 1. In situ reverse transcription/polymerase chain reaction.
Figure II, DNA-ISPCR of HIV-1-infected microtubule endothelial cell lines.