The plasmid isolated may not be correct. If you have that bacterial culture which showed PCR positive, try to make 1:100000 dilution, and spread 100 ul of the diluted culture on a fresh plate. When colonies grown, pick up 10-15 of them randomly, and make minipreps, and run both PCR and the restriciton digestion to screen pocitive clone.
The gel is commercial precasted one, if you use buffer other than manufactory recommended, the sample won't be stacked well.The other issue is that the sample wells seem not be rinsed thoroughly, and the sample loading could not be down to bottom of the well.Try to make fresh SDS-PAGE gel by yoursaelf for better Western blotting.
You need to run Western blot to probe if p65 expressed or not. When you prepare nuclear extracts, how many cells have been treated with TNF?If less than one million cells, next time increase to at least 5 millions. Make nuclear extracts using protocol not from the commercial kit, try to use your own methods, that means that you prepare everything k
It might be required for this lncRNA gene A product to stimulate gene B protein production, but it may not be necessary for this lncRNA functions as a resource to neutralize some miRNA that targets the gene B mRNA for RNAi. Is this lncRNA bound some miRNA or target gene B mRNA? Or is this lncRNA bound to DNA, or bound to some proteins?
According to the data, A is a lncRNA, and B is A target mRNA. It is make sense that A gene depletion causes downregulation of B, but B depletion shows no effect on A.The point is that how to demonstrate the lncRNA A regulates its target mRNA B, and why the lncRNA A is upregulated in drug-resistant, but is not in wild-type cell line.Does drug s