Making TE buffer for RNA disolution - afraid of RNase contamination (Feb/02/2005 )
We cannot use DEPC to treat TE buffer; so how do you prepare TE buffer for RNA disolution? Although I use DEPC-treated water to make TE buffer, but I am still afraid that RNase can contaminate through EDTA powder or while pH determination.
Any advice will be appreciated.
usually i don't use te buffer for rna dissolution.
Take care of every material used (pipette tips, falcon... ) and normally everything should be ok. Don't forget to store rna at -80° and keep them on ice every time. For dissolution you can make it at room temperature but take care of concentration of rna. If they are too concentrate, they probably form a gel.
I resuspend rna in FRESH DEPC treated water.
For EDTA, due to the fact you use DEPC treated water for dissolving it, sterilized material for dissolution and preservation, and sterilize edta solution (filtration onn 0.22µm filter), there will probably have no chance of an rnase contamination. Moreover if edta powder is well used, i mean never put back an excess of powder in the stock, there is probably no contamination.
For example, with good attention, i have an total RNA solution of very good quality, and have made it more tan a year ago !
I use as well just DEPC water for dissolving RNA.
Whilst working with RNA I use filtertips and clean my bench before with EtOH. Always worked fine and never had problems
just for completing my answer and marc_U7snurp's one, i wash my bench too with detegents and after with ethanol (to minimize ethanol induced bacterias' fixation on bench), and after i put a clean aluminium sheet.
For a good extraction, you'll need to be quick in the steps, in order to minimize time of extraction and risks of degradation. And try to be the most in ice.