Genomic DNA Isolation and Purification

Adapted from Invitrogen ChargeSwitch gDNA Plant Kit protocol: ChargeSwitch Plant gDNA

Using Ca. no. cs18000, lot no. 2130607

NOTE: This protocol is meant to isolate high molecular weight gDNA. To avoid shearing the DNA, do not vortex the purified DNA, avoid pipetting when possible and avoid creating bubbles during the isolation and purification process. If mixing is necessary, e.g. during genomic library preparation, mix by very gently flicking the tube and spinning down on a microfuge.

1. Prepare 2.5 mL Reagent A (300 mM CaCl2, 15% polyvinylpyrolidone) fresh as follows for plant samples rich in polysaccharides and polyphenolics.
   1. CaCl2 : 0.11025 g (2x=0.2205g)
   2. Polyvinylpyrolidone (10,000 average MW) : 0.375 g (2x=0.75g)
   3. ChargeSwitch® Lysis Buffer (L18) : 2.5 mL (2x=5mL)
   4. Mix well.
2. For Ganoderma lucidum samples double all noted reagent volumes through step 18.
3. To 900 μl ChargeSwitch® Lysis Buffer (L18), add 100 μl Reagent A and use this buffer to prepare samples (see next page). We will call this solution L18RA. 2x all noted reagent volumes for ganoderma lucidum
4. Chill the Precipitation Buffer (N5) on ice.
5. Harvest mycelium and spin down at 10k rpm, 10 minutes, 4C. Discard supernatant, wash by resuspending in 20 mL KCl/NaP or PBS or MilliQ H2O, pellet, repeat once more to end with a pellet of washed mycelium.
6. Between every sample: clean mortar and pestle by wiping with 10% bleach, rinsing with deionized water, then wiping with 70% ethanol, then rinsing with milliQ water.
7. Weigh 100 mg of wet mycelium, add the mycelium to mortar and add 1 mL of L18RA, freeze the mixture sample in liquid nitrogen and grind the frozen tissue to a powder using the mortar and pestle. Let liquid nitrogen evaporate from the sample and collect into a tube.
8. Let the sample thaw and then add 2 µL RNase A to each sample.
9. Vortex the mixture until completely resuspended.
10. Add 100 µL 10% SDS to 1 mL sample mixture. If you varied the amount of ChargeSwitch® Lysis Buffer (L18) in Step 3, ensure to maintain a ratio of 10:1 for ChargeSwitch® Lysis Buffer (L18) to SDS. Proceed with remaining steps in the manufacturer's suggested protocol.
11. Incubate at room temperature for 5 minutes.
12. Add 400 μl ChargeSwitch® Precipitation Buffer (N5) to the lysate. Mix by inversion or vortexing for 10 seconds until a precipitate forms. If different amounts of ml ChargeSwitch® Lysis Buffer (L18) are used, maintain a ratio of 10:4 for ChargeSwitch® Lysis Buffer (L18) to ChargeSwitch® Precipitation Buffer (N5).
13. Centrifuge at maximum speed for 5 minutes at room temperature to produce a clear lysate.
14. Transfer the clear lysate to a new, sterile 1.5 ml microcentrifuge tube for manual purification or a 96 x 2 ml deep well plate (page 15) for automated purification.
15. Proceed to Purification Procedure.
16. Thoroughly vortex the tube containing the ChargeSwitch® Magnetic Beads to fully resuspend the beads in the storage buffer.
17. Add 100 μl ChargeSwitch® 10% Detergent (D1) to the ∼l.2 ml lysate (from Step 10, page 7).
18. Add 40 μl resuspended ChargeSwitch® Magnetic Beads.
19. Mix gently by pipetting up and down 5 times using a 1 ml pipette tip set to 900 μl without forming any bubbles to evenly distribute the ChargeSwitch® Magnetic Beads within the solution. Important: Avoid forming bubbles by ensuring that the pipette tip is submerged during mixing, and by pipetting up and down gently.
20. Incubate at room temperature for 1 minute.
21. Place tubes on the MagnaRackTM (‘on the magnet’ position) until the ChargeSwitch® have formed a tight pellet.
22. Without removing the tubes from the magnet, carefully aspirate and discard the supernatant without disturbing the pellet of beads by angling the pipette such that the tip is pointed away from the pellet (see figure on page 5).
23. Proceed immediately to Washing DNA
24. Remove tubes from the magnet.
25. Add 1 ml ChargeSwitch® Wash Buffer (W12) to the tube and gently pipet up and down 5 times to resuspend the beads using a 1 ml adjustable pipette set to 900 μl to mix without forming bubbles.
26. Place tubes on the magnet until the beads have formed a tight pellet and the supernatant is clear.
27. Without removing the tubes from the magnet, carefully aspirate and discard the supernatant without disturbing the pellet of beads by angling the pipette such that the tip is pointed away from the pellet.
28. Remove tubes from the magnet.
29. Add 1 ml ChargeSwitch® Wash Buffer (W12) to the tube and gently pipet up and down 5 times to resuspend the beads using a 1 ml adjustable pipette set to 900 μl to mix without forming bubbles.
30. Place tubes on the magnet until the beads have formed a tight pellet and the supernatant is clear.
31. Without removing the tubes from the magnet, carefully aspirate and discard the supernatant without disturbing the pellet of beads by angling the pipette such that the tip is pointed away from the pellet.
32. Proceed immediately to Eluting DNA.
33. Remove the tube containing the pelleted magnetic beads from the magnet. There should be no supernatant in the tube.
34. Add 150 μl of ChargeSwitch® Elution Buffer (E6). Note: You may vary the elution volume (50-100 μl) to suit the application. Do not use volumes <50 μl for eluting DNA.
35. Pipet up and down gently 15-30 times using an adjustable pipette set to 100 μl (adjust to any variations on the ChargeSwitch® Elution Buffer volume used) until ChargeSwitch® Magnetic Beads are completely dispersed and no clumps are visible. Note: If a few grainy clumps remain, proceed to the next step. This will not adversely affect the result.
36. Incubate at room temperature for 1 minute.
37. Place tubes on the magnet until the beads have formed a tight pellet and the supernatant is clear.
38. Without removing the tubes from the magnet, carefully transfer the supernatant containing the DNA to a sterile microcentrifuge tube without disturbing the pellet by angling the pipette such that the tip is pointed away from the pellet.