Trizol for RNA extraction - (Feb/13/2008 )
Hi, everybody I'm new to this forum, and RNA extraction is the "First" experiment, so i'm really new and fresh to the research area, i don't know if this is an appropriate place to post this but here i go:
i did the lab today, i understand that Trizol agent contains many chemicals, like Guanidinium thiocyanate-phenol-chloroform extraction, how does these chemicals help during the extraction? i mean chemically... I know before that we have to freeze the neurospora with liquid nitrogen to inactive all the functions of the cells and lyse them, and grinding it into powder. So my question is what does these components do? Thank You
They lyse the cells and denature the RNases so they would chop up your RNA during extraction. Acid phenol extracts DNA/proteins and leaves the RNA in the aqueous phase. Chloroform helps the phase separation so it will be easier to recover the aqueous phase.
So basically, phenol traps DNA/proteins into organic phase (i thought proteins and DNA have charges and how can it be trapped in organic phase when phenol is hydrophobic?) , chloroform helps trapping lipid soluble substances? what about guanidinium? what does it do? and how does chloroform helps in the phase separation? how the pH affect the RNA and DNA separation which determines their ability to get into aqueous phase? thank you Sorry i'm really NEW to this thing.
sorry for this answer, but i think it is better for you to read about this in a molecular-bio book. there you will get the most satifiying answers for your questions.