TITLE: Guidelines for Using a Salt:Chloroform Wash to Clean Up HMW DNA for PacBio
AUTHORS: Miguel Flores

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol can be used to clean up high-molecular-weight genomic DNA (gDNA) prior to PacBio library preparation. It describes how to use a high-salt low-ethanol percentage wash to remove polysaccharides before DNA is precipitated from the solution.</div></div>

[STEPS]
?. Bring the volume of  HMW DNA up to 200 µL with Elution Buffer (EB) and label it as TUBE 1.
?. Add the following reagents to TUBE 1:  AB1DNA in EB  200 µL  2  5 M NaCl    100 µL  3  500 mM EDTA, pH 8  &nbsp;&nbsp;&nbsp;
  2 µL4  PacBio EB (10 mM Tris-HCl, pH 8.5)    198 µL  5  TOTAL    500 µL
AB1DNA in EB  200 µL  2  5 M NaCl    100 µL  3  500 mM EDTA, pH 8  &nbsp;&nbsp;&nbsp;
  2 µL4  PacBio EB (10 mM Tris-HCl, pH 8.5)    198 µL  5  TOTAL    500 µL
?. Prepare Buffer A and set aside:  AB15 M NaCl&nbsp;&nbsp;&nbsp;   &nbsp;100 µL  2  500 mM EDTA, pH 8    &nbsp;&nbsp;&nbsp;&nbsp;
  2 µL  3  PacBio EB (10 mM Tris-HCl, pH 8.5)  &nbsp;398 µL4  TOTAL   &nbsp;500  µL
AB15 M NaCl&nbsp;&nbsp;&nbsp;   &nbsp;100 µL  2  500 mM EDTA, pH 8    &nbsp;&nbsp;&nbsp;&nbsp;
  2 µL  3  PacBio EB (10 mM Tris-HCl, pH 8.5)  &nbsp;398 µL4  TOTAL   &nbsp;500  µL
500 µL of buffer will need to be prepared for each sample.
?. Add 400 µL of Phenol:Chloroform:Isoamyl Alcohol (25:24:1), pH 8 to TUBE 1.
?. Invert the tube 20 times to mix.
?. Spin the tube at maximum speed (at least 10 g) for 10 minutes at Room Temperature (RT).
?. Carefully remove the aqueous layer, do not disturb the interface. Place into a clean 2 mL microcentrifuge tube.Label the tube as TUBE 2.
?. Add 400 µL of Buffer A (from step 1) to TUBE 1.
?. Invert tube 20 times to mix.
?. Spin tube at maximum speed (at least 10 g) for 10 minutes.
?. Carefully remove the aqueous layer, do not disturb the interface. Place into TUBE 2.
?. . Measure the volume in TUBE 2: __________ µL. It should be close to 800 µL.
?. Add an equal volume of Chloroform:Isoamyl Alcohol (24:1) to TUBE 2.
?. Invert tube 20 times to mix.
?. Spin tube at maximum speed (at least 10g) for 10 minutes.
?. Carefully remove the aqueous layer, do not disturb the interface. Place into a clean 2 mL microcentrifuge tube. Label the tube as TUBE 3.
?. Measure the volume in TUBE 3: __________ µL.
?. Add 0.3X volume of ethanol (99.99%) to TUBE 3. This high-salt, low-ethanol mixture precipitates the excess polysaccharides while gDNA remains in the solution. _______ µl (TUBE 3) x 0.3 = _________ µL of Ethanol
?. Invert tube 20 times to mix.
?. Spin tube at maximum speed (at least 10 g) for 15 minutes.
?. Carefully remove the supernatant without disturbing the polysaccharide pellet. (Note that no visible pellet may be seen at this step). Place supernatant into a clean 2 mL microcentrifuge tube. Label the tube as TUBE 4 and measure the volume.
TUBE 4 contains the gDNA.
?. . Add 1.7X volume of ethanol (99.99%) to TUBE 4. The gDNA can be seen as falling out of the solution as long strands of gDNA; _______ µl (TUBE 4) x 1.7 = _________ µL of Ethanol.
If the final volume is larger than 2 mL, I recommend splitting the volume in two tubes (two tech reps).
?. Invert tube 20 times to mix.
?. Spin tube at maximum speed (at least 10 g) for 15 minutes. Discard supernatant with pipette, do not disturb the DNA pellet.
?. Add 500 µL of 70% ethanol to DNA pellet to remove the excess salt; do not disturb the DNA pellet.
?. Spin the tube at maximum speed (at least 10 g) for 15 minutes. Discard supernatant with pipette, do not disturb the DNA pellet.
?. Add 500 µL of 70% ethanol to DNA pellet to remove the excess salt; do not disturb the pellet.
?. Spin the tube at maximum speed (at least 10 g) for 15 minutes. Carefully remove the supernatant; do not disturb the DNA pellet.
?. Quick spin to gather the residual ethanol at the bottom of the tube and carefully remove with a P20 tip.
?. Let DNA pellet air dry for 5 min at room temperature, taking care not to over dry.
?. Resuspend the DNA pellet in 100 µL 10 mM Tris-HCl, pH 8. Incubate at 4ºC with gently mixing overnight to resuspend. Store at 4ºC for use within one week, or store at -80ºC for long-term storage.
If you have two technical reps, resuspend both DNA pellets with one round of 100 µL 10 mM Tris-HCl, pH 8.