TITLE: High-Molecular-Weight gDNA extraction protocol from moss gametophytes
AUTHORS: Lei  Zhu

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The purpose of this protocol is to extract high-molecular-weight genomic DNA from gametophyte tissue of the moss </span><span style = "font-style:italic;">Physcomitrella patens</span><span>, which contains lots of phenolic compounds. This protocol was modified from a CTAB method for DNA isolation from moss protonemal tissue (Ano et al., 2012). gDNA isolated using this modified CTAB protocol was pure, comparable to gDNA quality extracted from commercial extraction kits. Besides, the yield (69 μg/g FW) was relatively high. The high-quality gDNA is useful for PCR and Southern blotting for the detection of genome editing.</span></div></div>

[STEPS]
?. [Harvest Plant Materials]
Harvest 3-week-old moss gametophyte tissue.
?. [Harvest Plant Materials]
Carefully remove rhizoids from leafy gametophytes.
IMPORTANT: Rhizoids mixed in the green leafy tissue will cause pigment co-precipitated with DNA. Be careful to remove rhizoids from leafy gametophytes.
?. [Harvest Plant Materials]
Grind leafy gametophytes to fine powder in liquid nitrogen using the mortar and pestle.
IMPORTANT: Grinding moss leafy gametophytes to fine powder is very important. If the powder is coarse, the efficiency of extraction will be much lower than expected.
?. [Harvest Plant Materials]
Transfer the ground powder to a  conical tube.
50 mL
?. [Harvest Plant Materials]
Put the conical tube in a  freezer with cap open until all liquid nitrogen evaporate.
-80 °C
?. [Crude Extraction in CTAB Buffer]
Add  pre-heated 2x CTAB buffer to the  conical tube that contains ground gametophyte powder.
10 mL
50 mL
?. [Crude Extraction in CTAB Buffer]
Close the cap and shake the tube vigorously to mix CTAB buffer and gametophyte powder.
?. [Crude Extraction in CTAB Buffer]
Incubate the conical tube in  water bath for .
65 °C
?. [Crude Extraction in CTAB Buffer]
Shake the tube to mix buffer and gametophyte powder every .
?. [Crude Extraction in CTAB Buffer]
Take the conical tube out of the water bath and cool down to .
65 Room temperature
?. [Crude Extraction in CTAB Buffer]
Add  chloroform: isoamyl alcohol to the conical tube. Mix well.
10 mL
?. [Crude Extraction in CTAB Buffer]
Then put the tube to a rotator for  at .
65 Room temperature
?. [Crude Extraction in CTAB Buffer]
Centrifuge at  for  at  to separate phases.
Centrifuge: 1000 34
65 Room temperature
?. [Crude Extraction in CTAB Buffer]
Transfer the upper supernatant to a new 50 mL conical tube. Add  10% CTAB and  chloroform: isoamyl alcohol to the new tube. Shake well.
1 mL
10 mL
?. [Crude Extraction in CTAB Buffer]
Incubate the conical tube at  for .
65 Room temperature
?. [Crude Extraction in CTAB Buffer]
Centrifuge the conical tube at  for  to separate phases.
Centrifuge: 1000 34
?. [Crude Extraction in CTAB Buffer]
Transfer the upper aqueous supernatant to a new conical tube. Add  isopropanol and mix well.
10 mL
?. [Crude Extraction in CTAB Buffer]
Centrifuge the conical tube at  for  to precipitate the pellet.
Centrifuge: 1000 34
?. [Crude Extraction in CTAB Buffer]
Decant the supernatant. Air dry the pellet.
?. [Crude Extraction in CTAB Buffer]
Dissolve the pellet in  TE buffer.
4 mL
Tap the pellet gently until it dissolves completely.
?. [Crude Extraction in CTAB Buffer]
Centrifuge the tube at  for .
Centrifuge: 1000 34
?. [Crude Extraction in CTAB Buffer]
Transfer the supernatant to a new   tube. Discard pellets.
50 mL
?. [Crude Extraction in CTAB Buffer]
Incubate the tube  for .
on ice
?. [Crude Extraction in CTAB Buffer]
Centrifuge the tube at  for  to precipitate the pellet.
Centrifuge: 1000 34
?. [Crude Extraction in CTAB Buffer]
Rinse the pellet with 70% ethanol. Centrifuge the tube and discard the supernatant.
?. [Crude Extraction in CTAB Buffer]
Air dry the pellet. Dissolve the pellet in  TE. Transfer DNA in TE to a new   Eppendorf tube.
700 µl
1.5 mL
Tap the tube gently. Wait until the pellet is completely dissolved. DNA can be stored at this step for overnight at  .
?. [Crude Extraction in CTAB Buffer]
Add RNase A into DNA solution at a final concentration of . Mix well.
?. [Crude Extraction in CTAB Buffer]
Incubate the Eppendorf tube at   for .
37 °C
?. [Removal of protein]
Add  Tris-saturated Phenol. Mix thoroughly.
700 µl
?. [Removal of protein]
Centrifuge at  for  at  to separate phases.
Centrifuge: 16.000 1324
37 Room temperature
?. [Removal of protein]
Transfer the upper phase to a new  Eppendorf tube. Leave white stuff in the middle layer between two phases. Discard the middle and lower phases.
1.5 mL
?. [Removal of protein]
Repeat steps 33-35 to extract DNA in phenol one more time.
?. [Removal of protein]
After transferring the upper supernatant to a new tube, add an equal volume of phenol: chloroform to the new tube. Mix thoroughly.
?. [Removal of protein]
Centrifuge the Eppendorf tube at  for  at  to separate phases.
Centrifuge: 16.000 1324
37 Room temperature
?. [Removal of protein]
Transfer the upper phase to a new Eppendorf tube. Discard the pellet.
?. [Removal of protein]
Centrifuge the tube at  for  at  to separate phases.
Centrifuge: 16.000 1324
37 Room temperature
?. [Removal of protein]
Repeat steps 40-42 once to remove the phenol residue.
?. [Precipitation of Genomic DNA]
Add 1/10 volume of  sodium acetate and 2x volume of pre-chilled 100% ethanol to the supernatant. Mix well.
?. [Precipitation of Genomic DNA]
Incubate the tube   for .
on ice
?. [Precipitation of Genomic DNA]
Centrifuge the tube at  for  to collet the pellet. Discard the supernatant.
Centrifuge: 16.000 1324
?. [Precipitation of Genomic DNA]
Centrifuge the tube at  for . Discard the ethanol.
Centrifuge: 16.000 1324
Pellet may not be attached tightly to the bottom of the Eppendorf tube. Be careful to pour off the supernatant.
?. [Precipitation of Genomic DNA]
Air-dry the pellet by leaving the Eppendorf tube in the fume hood. Wait until ethanol residue evaporated, but not over-dry the pellet.
?. [Precipitation of Genomic DNA]
Dissolve the pellet in  TE. Tap to dissolve genomic DNA.
100 µl
?. [Harvest Plant Materials]
Weigh – fresh weight of leafy gametophytes.
2 g
5 g
?. [Harvest Plant Materials]
Pat dry leafy gametophytes with clean towel papers.
?. [Removal of protein]
Add  chloroform: isoamyl alcohol to the new tube. Mix thoroughly.
700 µl
?. [Removal of protein]
Transfer the aqueous supernatant to a new  Eppendorf tube.
1.5 mL
?. [Precipitation of Genomic DNA]
Add  70% ethanol to wash the pellet. Finger tap the tube to mix.
1 mL
?. [Crude Extraction in CTAB Buffer]
Incubate the conical tube  for .
on ice
?. [Crude Extraction in CTAB Buffer]
Add  sodium acetate and  100% pre-chilled ethanol to the new tube. Mix well.
400 µl
8 mL
?. [Precipitation of Genomic DNA]
Store extracted genomic DNA in the fridge until use.
Additional Notes:1. The entire procedure takes about 4 hours.2. The yield and quality of gDNA from four preps are listed below. FW, fresh weight. Table 1. The yield and the quality of genomic DNA.Gametophyte Tissue Extraction Method Yield (μg/g FW) OD 260/280 OD 260/230WTThis method69.21± 8.43 1.95 ± 0.03 2.21 ± 0.12WTNucleoSpin PlantDNA Extraction Kit5.81± 0.70 1.99 ± 0.02 2.20 ± 0.05 3.