Editing Genomic DNA from Plants (section)

=== <span style="line-height: 20.8px">Protocol</span> ===

<br/><span style="line-height: 20.8px">'''Preparatory steps'''<br/>Before grinding, pre-chill the mortar and pestle (to minimize frozen tissue thawing) and 95% ethanol solution at -20°C. Pre-heat water baths (65°C and 37°C) before beginning the extraction. Once pre-heated, prepare 10 mL (per 1 g of leaf tissue) extraction buffer by adding 0.3% (v/v) β-mercaptoethanol in a 50 mL Falcon tube, and pre-heat in the 65°C water bath. PVP can also be added at this point, but is not required.</span>

<span style="line-height: 20.8px">'''Grinding and tissue disruption'''<br/>Using liquid nitrogen, grind 1 g of frozen leaf tissue into a fine powder. Place the powder into a new 50 mL Falcon tube and mix in the pre-heated extraction buffer. Put the sample into the 65°C water bath and mix by inversion every 10 min for 30 min- 1 h. After incubation, centrifuge the sample tube for 5 min at 5000 × g (to pellet and remove un-lysed leaf tissue) and decant the supernatant into a new 50 mL Falcon tube.</span>

<span style="line-height: 20.8px">'''Protein Extraction and RNAse treatment'''<br/>Add 1 volume of chloroform: isoamyl alcohol to the solution and mix by inversion for 5 min. Centrifuge the sample for 10 min at 5000 × g and pipette the upper aqueous phase into a new Falcon tube, taking care to avoid the aqueous/organic layer interface. Add 5 μL of RNAse A (10 mg/mL) to the solution and incubate at 37°C for 15 min with periodic, gentle mixing. After incubation, add 1 volume of chloroform: isoamyl alcohol to the solution and mix by inversion for 5 min. Centrifuge the solution for 10 min at 5000 × g and pipette the aqueous phase into a new Falcon tube, again taking care to avoid the organic layer.</span>

<span style="line-height: 20.8px">'''Precipitation'''<br/>Add ½ volume of 5 M NaCl to the sample and mix gently by inversion. Then, add 3 volumes of cold 95% ethanol and mix gently by inversion. Place the tubes into a -20°C freezer and incubate for 1 h. NOTE: do not leave the sample at -20°C for more than 1 h as both the CTAB and NaCl can precipitate from solution, preventing DNA isolation.</span>

<span style="line-height: 20.8px">After incubation, centrifuge the Falcon tube for 10 min at 5000 × g to pellet the DNA. Carefully decant away the supernatant and wash the DNA pellet with 3 mL of 70% ethanol. Gently swirl the solution and centrifuge again for 10 min at 5000 × g. Carefully decant the supernatant and air-dry DNA pellet for 15 min at room temperature. Once dried, suspend DNA in 200 μL of TE buffer.</span>

<span style="line-height: 20.8px">'''DNA quality and quantity assessment'''<br/>Assess the quality of the extracted DNA using a NanoDrop UV/Vis spectrophotometer and 0.7% (w/v) agarose gel, looking for a single absorbance peak at 260 nm, a 260/280 absorbance ratio of 1.8-2.0, and no evidence of substantial band shearing or contamination (either RNA or polysaccharide).</span>
[[Category:Protocols]]