Problem 1: In my research, I encountered an issue with my wet transblot procedure: despite using 8-10ug of RNA sample and applying a voltage of 10 v for 2 hours in TBE transfer buffer, I observed incomplete transfer of the top band from the UREA gel (8M) to the nylon membrane upon examination.
Problem 2: for northern blot, I conducted prehybridization at temperatures ranging from 55°C, 60 °C, and 65°C, followed by membrane washing with SSC buffer and blocking with blocking solution. Subsequently, I proceeded wash the membrane in wash buffer and soak in detection buffer and applied CDP-Star on top of the membrane. The entire membrane exhibited fluorescence (is it normal?), later the resulting X-ray film exposure did not reveal the desired bands. Background noise quite bad. I would appreciate any professional guidance or suggestions to address this discrepancy."
problem in northern blotting is often sample degradation by RNases (both endogenous to the sample and through environmental contamination), which can be avoided by proper sterilization of glassware and the use of RNase inhibitors such as DEPC (diethylpyrocarbonate)
Northern blots are used to detect the presence of specific mRNA molecules. To do a northern blot, RNA is loaded into the wells of a gel, and separated according to size by electrophoresis. The RNA is then transferred to a membrane filter in a process called blotting.
Northern Blots: 0.05 fmol detection limit with near-IR RNA Probes.
To disrupt the secondary structure of RNA, either formaldehyde or glyoxal/DMSO (dimethyl sulfoxide) is commonly used as a denaturing reagent. Formaldehyde is simply added to the samples and gels, so that the method using formaldehyde is easier to run the gels than that using glyoxal/DMSO.
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