A B. distachyon leaf was frozen using liquid nitrogen and then ground up. DNA extraction buffer (DEB) was added. DEB contains 1% sodium dodecyl sulfate (SDS), a detergent which binds and solubilizes lipids found in the cell membrane. DEB also contains 25 mM ethylene diamine tetra-acetic acid (EDTA), which chelates metal ions that enzymes, such as DNases, require to function, thereby inhibiting their function. DEB also contains b-mercaptoethanol, a disulfide bond cleaver to further inhibit protein function [1]. Potassium acetate (KOAc) was used to precipitate proteins and carbohydrates while nucleic acids remained soluble. 100% isopropanol was used to precipitate the nucleic acids from the salty solution. The pellet was rinsed with 70% ethanol to remove sodium. The pellet was resuspended and stored in 50 mL T10E1 buffer, which contains 10 mM Tris and 1 mM EDTA.
The 50 uL of solution was split evenly, and one sample was digested with RNase A, an endonuclease that cleaves the phosphodiester bonds of single stranded RNA at C and U residues [2]. ½ dilutions of both RNase treated and RNase untreated DNA were prepared using 10 mM Tris. DNA samples were prepared for gel electrophoresis using glycerol, Bromophenol blue, and Xylene cyanol. Glycerol is a heavy molecule that ensures DNA sinks in the gel wells. Bromophenol blue migrates faster than Xylene cyanol, which allows visualization to ensure the samples run adequately but not off the gel [5]. A 0.9% agarose gel was run at 100 volts for 30 minutes. Agarose forms pores suitable for size separation of nucleic acids [3]. SYBR Safe DNA Gel Stain was used in the gel as a safe alternative to ethidium bromide [4].
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