I had extracted total RNA of cells according to the manufactures and tried to extract DNA of the same sample respectively, but i see no bound on electrophoresis gel. I followed all steps one by one as protocol says but still I can not find DNA fragmentation ladder( these are apoptotic cells). any one please help me to find the errors. one more thing, I have stored the samples in -80 degrees of celcius after extracting total RNA and now after 2 months it's impossible to extract DNA from phenol-ethanol phases.
You can extract the DNA from phenol-ethanol phase due to the recommendation of Trizol reagentleaflet . Wile you extract the DNA, their are many errors that can happened in several step for example insufficient volume of Trizol reagent, other reagent or sample as well(type, period of stored sample). I'm use to extract the DNA from clotted blood which stored -80 celcius more than 2 years and I got very good yield and purity of the DNA . You might lost the DNA in washing step (the way to discard the supernatant or air dry of the DNA pellet if this step had completely dry your DNA would hardly to dissolve). Mixing step is critical too, for washingstep you should mix the supernatant really very gentle mixing and too vigorously mixing may shear the DNA.
Check pH of aquous and organic phase. It has to be acidic! Increment ethanol volumen in order to assure DNA to precipitate from organic phase.
Did your RNA extraction procedure successful? Did you see any band on the gel after amplification? if you have problem with DNA only, maybe your problem is your cells, you said they are apoptotic cells and one stage of apoptosis process is genome degradation, maybe your cell's genome has degraded in apoptosis process
Dear Mojtaba Rasti
Yes I did.
exactly it's my problem... if they are apoptotic it means that I have to see DNA ladder fragmentation on gel but there is nothing ...
best regards
Change your extraction procedure, use column system to extract DNA. maybe you are losing your DNA fragments cause of their short length in Trizol process.
yours
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