Handling RNA - How to avoid RNase contamination
Posted 02 December 2010 - 09:19 PM
Posted 26 May 2011 - 03:23 AM
something that you might consider for RNA extraction is using liquid nitrogen:
-In a mortar, where i previously flamed 100% ethanol, washed with RNAse Zap equivalent, flash freeze the tissue.
Then smash the tissue with the pestle (also treated with RNAse Zap) into powder.
-pour the powder into a tube where you add the trizol.
-follow normal protocol (trizol-chlorophorm).
I usually keep adding a little bit of liquid nitrogen in the mortar while smashing to make sure my tissue sample remains frozen. This technique is truly efficient since the tissue is well preserved.
Hope this will help some of you
Posted 10 June 2011 - 04:17 PM
We usually prepare 4x 50 mL tubes. Fill three with 40 mL DEPC treated water, and one with 100% ethanol.
One of the DEPC treated water tubes gets 5-10 squirts of RNase Zap.
Between each sample, the homogenizer is dipped into 100% ethanol, RNase Zap water, and 2x DEPC treated water in turn.
Each time turning the homogenizer on for about 15-30s.
This is fairly quick and seems to work well for us.
I do exactly like that. The difference is that a fill one tube ( 2ml) with 100% ethanol, one with treated water and RNAzap squirts and another one just with treated water. I prepare these three tubes for each sample. So, after the homogenizing I wash the tip of the homogenizer on these tubes in that sequence. The next sample I change tubes and use new ones. My concern is avoid cross-contamination. Like they said before, the tissue is full of RNAse!