I have been trying to isolate RNA from C. elegans by the trizol method, but when I go to the quatification step (with biodrop) the sample has no RNA. I imagine the trouble can be with the lysate step - I'm putting the tube with worms and trizol (1mL) in liquid nitrogen, let thaw at 37 °C, and repeat several times (3 – 6 x). Then, I follow the trizol protocol; add 0,2 ml of chloroform and so on.
Does anyone have any tips or suggestion to improve my protocol; or a different protocol to isolate RNA from C. elegans?
Thank you
Try adding in a proteinase K digestion. Take out the tubes & mix every 10-15 minutes for about an hour.
I have used trizol kit of H. armigera. i first crush the worm in liquid nitrogen and then add trizol reagent.
Are you removing all the M9 buffer? We would rinse and let settle by gravity with M9 three times, remove all M9, flash freeze the worms and add Trizol directly to the tube. Also, you do not need so much Trizol. I would say 250ul would be enough, then adjust your chloroform amount since you are using less Trizol.
Typically, you will need liquid nitrogen and ground the worms and immediately add your solution (e.g., Trizol) before thawing. You could use the boiling method, but you will check to see which method would work better in your hands.
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