DNA blotting and narrow line blotting experiments
DNA blotting and narrow line blotting experiments
Spot and narrow line blotting experiments that can be used to detect the relative abundance of target sequences in blotted 1DNA products. The main applications are (1) genetic disease diagnosis, (2) DNA profiling, (3) detection of DNA and its content in samples, and (4) PCR product analysis.
Operation method
multifractionation
Principle
Spot and narrow line blotting is a technique in which mixed, unseparated surfaces are set on a nitrocellulose or nylon membrane for hybridization analysis.
Materials and Instruments
DNA Move 1. Cut a nylon membrane the same size as the Multi-Filtration Sampler and place the membrane on the surface of the 6xSSC and allow it to submerge naturally. Allow to stand for 10 min.2. Cut a piece of Whatman 3 MM filter paper the same size as the Multifilter Sampler and wet it with 6xSSC.3. Place the Whatman 3 MM filter paper on the Multi-Filtration Sampler and place the membrane on top of it. Install the Multi-Filtration Sampler according to the manufacturer's instructions, making sure there are no air leaks in the unit. Caveat The positive reaction on the membrane is in the form of a band. The following issues should be noted in the experiment: the membrane transfer must be sufficient to ensure that the DNA has been transferred to the membrane. Hybridization conditions and rinsing are the key to ensure a good contrast between the positive result and the background. Insufficient rinsing will lead to too dark a background, while excessive rinsing may lead to false negatives. If toxic substances are used, environmental protection and safety must be observed. Common Problems Main applications 1. Genetic disease diagnosis 2.DNA profiling 3.Detection of DNA and its content in samples 4.PCR product analysis For more product details, please visit Aladdin Scientific website.
SSC NaCl NaOH Tris-Cl
Ultraviolet transilluminator Electrophoresis instrument Multi-filter sampler
4. Add 20xSSC to the DNA to give a final concentration of 6×SSC in a volume of 200~400 ul, denature at 100°C for 10 min, and place on ice.5. Connect the pipetting device with the Multi-Draw Sampler and add 500 ul of 6xSSC to each well.6. Centrifuge the DNA samples for 5 s, add the samples into each well, be careful not to let the pipette tip touch the membrane, and let the samples filter.
7. Disassemble the Multi-Filtration Sampler unit and place the membrane on a piece of Whatman 3 MM filter paper soaked in denaturing solution and allow to stand for 10 min.8. Transfer the membrane to a piece of Whatman 3 MM filter paper soaked in neutralizing solution. Leave for 5 min. 9.
9. Place the membrane on a sheet of Whatman 3 MM filter paper and allow to dry.10. Place the nylon membrane in a UV-transparent plastic sandwich and expose the DNA side down to a UV transilluminator for an appropriate amount of time to immobilize the DNA.11. Place the dried membrane between Whatman 3 MM filter papers and store at room temperature for several months.