Hi, i´m trying to isolate RNA from 20 days Cavia porcellus embryos. Today i tried the following protocol:
– Homogenize the tissues (the whole embryo in this case, not larger than 5 mm I think) with 300 uL RNA-solv reagent (on a 1.5 mL microcentrifuge tube)
– After homogenization, added 40 uL of clorophorm RNAase free
– Vortex 15 sec
– 10 min incubation on ice
– Centrifuge at 12.000 x g for 15 min at RT
– Took the aquose phase (clear, upper phase) and transfered to another tube
– Added 100 uL of Isopropilic alcohol for precipitation
– 10 min incubation at RT
– Centrifuge at 12.000 x g for 10 min at RT
– Discarded the supernatant and washed the pellet with Etanol 80% RNAase free
– Centrifuge at 7.500 x g for 5 min at RT (*)
– Discard Etanol, wait for tube to dry
– Add H20 RNAase free
It was everything ok, but when I made the last centrifugation (marked with a (*) the pellet just DISSAPEARED!! I tried repeating centrifugation at higher speed, but even so didn´t see a single white on my sample. Previous to washing the sample with Etanol 80%, there was a good amount of pellet. When do you think i made the mistake?
I do not think it is a mistake.It might be just invisible. I do not know about your situation but for example after LiCl precipitation, pellet is ideally invisible.
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