I tried to isolate RNA from trizol and triton x methods but I couldn't do it.
what happened with the trizol method? have you sucessfully isolated any rna with trizol method, if yes i dont need to tell you what steps can degrade the rna.. if no, then a few options., firstly familairise with trizol extractions rna sensitive parts, change incubation times and spin down to maximise your rna yield. what happened with the extraction. if you did not see any rna at all, try higher cell density or better cell lysis incubation, check if the reagent is active and not too old or phase seperated. if you did see rna but degraded then do it with more stringent conditions, use rnazap if you can. also there is a trizol bacterial rna extraction kit (https://www.lifetechnologies.com/order/catalog/product/16096040) , may be you dont want to use kits but possibly some procedural difference comapred to human tissue in this might help you as i have experience only with human tissue. hope it helps.
Hello Prince, problem is there is no pellet formation after precipitation step, and when run it on the gel a smear is formed no bands.
so if you have done it before you will know that the pellet is usually very delicately attached to the bottom than most other pellets we obtain in most procedures. my pellet has sometimes dislodges by the action of just inserting the pipette tip, so one tip is spin it down with the hinge of cap facing away from the centrifuge's center, in that way you are to expect the pellet at tube part of the bottom towards the hinge of the tube. also the pellet can be very small, before removing precipitate, check where it is, so as to not to dislodge it. the pellet could be white but also colourless and gel like in some cases. you can try overnight precipitation, and/or inert coprecipitant (glycogen, yeast rna, linear acrylamide) could help. may be use more cells. degradation could be an issue so care more. basically, longer incubation, more careful with the dislodging and then missing it, may be precipitation agent , longer spin down. let me know if any of this works.
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