I am extracting RNA from barley leaves and my gel photos are all like these ones. The problem is that other people used the same method for this plant and they look to not have a problem with that. I have tried to be more cautious about cleanness and RNAse issue, but it did not help as well. Do you have any idea what the problem can be?
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Barley leaves (snap-frozen) were crushed in a microcentrifuge tube using a sterilized metal rod. Approximately 100 mg of the crushed tissue was mixed with 1 mL of TRIsure and incubated at room temperature (RT) for 5 minutes. 200 μL of 24:1 chloroform: isoamyl alcohol was added to the above suspension and the tube was shaken vigorously for 15 seconds and then incubated at RT for 3 minutes. The mixture was centrifuged at 4⁰C at 14,000 xg for 15 minutes and the colourless supernatant was transferred to a sterile microcentrifuge tube containing 500 μL of isopropanol. The tube was held at RT for 10 minutes and then centrifuged at 4⁰C at 14,000 xg for 10 minutes. The RNA pellet so formed was washed with 75% ethanol (made with DEPC-treated water) and centrifuged at 4⁰C at 4,000 xg for 5 minutes. The pellet was air-dried and then dissolved in 33 μL of DEPC-treated water (Sambrook and Russell 2001).