RNase monster - how to avoid RNA degradation? - (Dec/08/2006 )
i want to know how equipments ,like gloves,blade and sicissors and surroundings can be sterilize against RNAse activity to safely isolate the rna.
my superviser says autoclave glass ware and gloves and heat sterilize aluminium foil,and tips etc.
we also dont have liquid nitrogen and the tissue sample is also very smal weights 13mg.
would i b successful in isolating mRN for pcr.
i have DEPC water ,is this be enough for inhibiting rnase activity.
i will be thankfull and waiting for suggestions and guidance.
I use to cleaned all with rnase zap (ambion), rinse with DEPC waterand autoclave glass, metals etc. For the sample ambion have buffers that will mantein your sample with out need to have it in LN2.
well sterilization of RNAse solution is done by autoclaving sometimes....
Better is to use guaranteed new tips RNase free, tubes etc...
Vessel can not be guaranteed so i rinse it by 0.1N NaOH (i use that solution several times) and rinse in DEPC water. Empty he bottle and then autoclaving the glassware.
Finally work at cold and quick.
i isolated very little quantity of rna.
i solubalizes it in autoclaved distilled water.not DEPC treated.
is it correct.
can i use depc treated water for solubilization and storage.does it interfere with reverse transcription pcr.
what is preferable autoclaved distilled water or depc treated water for solubilization or .5% SDS solution.
thanks for reply
RNA will dissolve in both DEPC treated and distilled water... DEPC degrades in the presence of water, heat and pressure to form CO2 and water therefore the treated water should be essentially equivalent to distilled (BTW we use DEPC treated MilliQ water for RNA) only it should be RNase free. DEPC treated water should not interfere with downstream steps, however occasionally a batch that hasn't quite been prepared properly will give some funny results with RT-PCR.
Always use DEPC treated water for the RNA, RNases are very tough and could easily be active in your MilliQ system. I have never heard of using a 0.5% SDS solution, but that would interfere with further reactions.