The construct is a mammalian cell overexpressing vector. There are two expressing cassettes: one is CMV promoter-MCS-SV40 poly A, the other one SV40 promoter-Neomycin resistant gene (Neo)-TK poly A. The CMV-MCS-SV40 poly A cassette is for testing cloned transgene expression. The transgene can be in Flag N-terminal tag fusion, and the transgen
Digitonin extraction bufferDigitoninEDTA (0.2 mM)HClPMSF (phenylmethylsulfonyl fluoride)PIPES buffer (4X)Dissolve 18.75 mg of digitonin by boiling with 10 mL of 4X PIPES buffer in a small flask with a stir bar. Add 1 mL of 100 mM PMSF while the buffer is still warm. Combine with 15 mL of
It should be a process to isolate viral DNA (small sized DNA, 2-20 kb) out of a mixture containing really large sized genomic DNA (50-500 kb). For genomic DNA, it is not necessary to isolate it from the mixtures with viral DNA, it is very simple to isolate genomic DNA from a tissue culture without any viral infection.
miRNA functions when it associates with specific target mRNA. When the targeted mRNA is in the process of degradation, reverse transcription could be affected due to the template can not be primered or primered cDNA can not be extented in reverse transcription. However, the inhibition is not from direct action by miRNA. So far, there is no direct e