TITLE: CTAB genomic DNA-extraction
AUTHORS: Michael  Burgis

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for the isolation of genomic DNA for a wide range of plant species.</div><div class = "text-block">This protocol is based on a paper published by Aboul-Maaty et al. 2019 (https://doi.org/10.1186/s42269-019-0066-1).</div><div class = "text-block">Comments have been added in bold in brackets.</div><div class = "text-block"><span>CTAB is used here to filter out phenolic compounds usually found in plant samples that cannot be removed otherwise. We tried extracting DNA using established protocols for </span><span style = "font-style:italic;">C.reinhardtii</span><span> although this doesn't work that well and the quality of the sample is very poor.</span></div></div>

[STEPS]
?. [Buffer preparation]
3X Extraction buffer (autoclaved): 3% CTAB (w/v), 1.4M NaCl, 0.8M Tris-HCl pH 8.0, 0.5M EDTA pH 8.03M Potassium acetate6M NaCl1x TE Buffer (autoclaved): 10mM Tris-HCl pH 8.0, 1mM EDTA pH 8.0
?. [Grinding plant sample]
Preheat 3x Extraction buffer in water bath at 65°C. Add 0.3% 2-β-mercaptoethanol to the 3× CTAB extraction buffer immediately before use.
?. [Grinding plant sample]
Grind 50 mg of plant samples into powder in liquid nitrogen using pre  chilled mortar and pestle. While still in the mortar, add 800 μl of the  preheated 3× CTAB extraction buffer to the grinded plant samples and  swirl gently to mix using the pestle. (This can be scaled depending on how much plant sample is used)
?. [Grinding plant sample]
Transfer the sample mixture to a 2-ml microcentrifuge tube, incubate in  water bath at 60–65 °C for 1 h, mix gently every 20 min by inverting the  tube for 20 times each, then cool down to the room temperature. (If using greater volumes it is recommended to switch to either 15ml or 50ml falcon tubes)
?. [Chloroform washing]
Add an equal volume of chloroform:isoamyl alcohol (24:1 v/v) and mix by slight inversion.
?. [Precipitation + Washing of DNA pellet]
Estimate the volume of the aqueous phase (approximately 700 μl) then add half this volume (350 μl) of 6 M NaCl and mix well. Successively, add  1/10 the volume (70 μl) 3 M potassium acetate and simultaneously mix with 500 μl ice-cold 100% isopropyl alcohol (approximately two-thirds the volume of the aqueous phase). Invert gently to precipitate DNA until the formation of DNA threads.
?. [Chloroform washing]
Centrifuge at 13,000 rpm for 15 min at room temperature (RT).
?. [Chloroform washing]
Using a wide-bore pipet, carefully transfer the upper aqueous phase, which contains the DNA, to a new 1.5-ml Eppendorf tube.
?. [Chloroform washing]
Repeat steps 5-8 until the upper aqueous phase is clear.
?. [Precipitation + Washing of DNA pellet]
Incubate at − 20 °C for 30 min.
?. [Precipitation + Washing of DNA pellet]
Centrifuge at 13,000 rpm for 5 min, discard the supernatant.
?. [Precipitation + Washing of DNA pellet]
Invert the tube containing the DNA pellet on tissue paper to complete draining off the supernatant.
?. [Dissolving and Analysis of DNA]
Resuspend the DNA pellet in 50 μl 1× TE buffer. Incubate the DNA at 50 °C for 1 to 2 h to ensure complete re-suspension. (I usually did this overnight to increase dissolving time)
?. [Precipitation + Washing of DNA pellet]
Wash the DNA pellet with 500 μl of 70% ethanol and invert once (to dissolve residual salts and to increase the purity of the DNA).
?. [Precipitation + Washing of DNA pellet]
Centrifuge at 13,000 rpm for 5 min.
?. [Precipitation + Washing of DNA pellet]
Discard 70% alcohol from tubes. invert the on filter paper, and allow tubes containing pellet to air dry at room temperature for 15 min. (Incubating at 60°C worked fine and saves time)
?. [Dissolving and Analysis of DNA]
Measure the quality and quantity of your DNA using a NanoDrop. (Usually, I get around 25µg of genomic DNA from 50mg of plant sample)