3. Homogenize using a tissue lyser for 60 s at 30 oscillations s
À1 .
4. Repeat step 3.
5. Add 50 μL of extraction buffer.
6. Mix by vortexing.
7. Transfer to PCR tubes (eight-strip format or 96-well plate).
8. Place in a thermal cycler and use the following parameters:
95
C for 10 min and 4
C for 5 min.
9. Add 50 μL of 3% (w/v) BSA.
10. Mix by vortexing.
11. Centrifuge at 1000 Â g for 15 min.
12. Collect the supernatant, which contains the gDNA.
3.5.2 T-DNA Integration
Analysis
1. Set up a PCR using Cas-specific primers and the extracted
gDNA in order to select (a) hairy root and primary plant
transformant (T0) lines in which the T-DNA is present or
(b) plant T1 and T2 lines in which the T-DNA is absent (see
Note 41).
3.5.3 Gene Mutagenesis
Analysis
1. Set up a PCR using the primers designed in step 3 of Subheading 3.1.3 to amplify the gRNA(s) target region(s) in the
extracted gDNA (a) from hairy root and primary plant transformant (T0) lines containing the T-DNA or (b) from plant T1
and T2 lines not containing the T-DNA (see Note 42).
2. Mix the magnetic beads for PCR amplicon purification by
vortexing to resuspend the magnetic beads.
3. Add 9 μL of magnetic beads for PCR amplicon purification to
5 μL of PCR reaction (see Note 43).
4. Mix by pipetting.
5. Incubate at room temperature for 5 min.
6. Place on the magnetic separator (see Notes 44 and 45).
7. Remove the cleared supernatant (see Note 46).
8. Add 200 μL of 80% ethanol.
9. Incubate at room temperature for 30 s.
10. Remove the cleared supernatant (see Note 46).
11. Repeat steps 8–10.
12. Incubate at room temperature for 10–15 min to dry the beads
(see Note 47).
13. Remove from the magnetic separator.
14. Add 50 μL of 10 mM Tris–HCl.
15. Mix by pipetting.
16. Incubate at room temperature for 2 min.
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Gwen Swinnen et al.
À1 .
4. Repeat step 3.
5. Add 50 μL of extraction buffer.
6. Mix by vortexing.
7. Transfer to PCR tubes (eight-strip format or 96-well plate).
8. Place in a thermal cycler and use the following parameters:
95
C for 10 min and 4
C for 5 min.
9. Add 50 μL of 3% (w/v) BSA.
10. Mix by vortexing.
11. Centrifuge at 1000 Â g for 15 min.
12. Collect the supernatant, which contains the gDNA.
3.5.2 T-DNA Integration
Analysis
1. Set up a PCR using Cas-specific primers and the extracted
gDNA in order to select (a) hairy root and primary plant
transformant (T0) lines in which the T-DNA is present or
(b) plant T1 and T2 lines in which the T-DNA is absent (see
Note 41).
3.5.3 Gene Mutagenesis
Analysis
1. Set up a PCR using the primers designed in step 3 of Subheading 3.1.3 to amplify the gRNA(s) target region(s) in the
extracted gDNA (a) from hairy root and primary plant transformant (T0) lines containing the T-DNA or (b) from plant T1
and T2 lines not containing the T-DNA (see Note 42).
2. Mix the magnetic beads for PCR amplicon purification by
vortexing to resuspend the magnetic beads.
3. Add 9 μL of magnetic beads for PCR amplicon purification to
5 μL of PCR reaction (see Note 43).
4. Mix by pipetting.
5. Incubate at room temperature for 5 min.
6. Place on the magnetic separator (see Notes 44 and 45).
7. Remove the cleared supernatant (see Note 46).
8. Add 200 μL of 80% ethanol.
9. Incubate at room temperature for 30 s.
10. Remove the cleared supernatant (see Note 46).
11. Repeat steps 8–10.
12. Incubate at room temperature for 10–15 min to dry the beads
(see Note 47).
13. Remove from the magnetic separator.
14. Add 50 μL of 10 mM Tris–HCl.
15. Mix by pipetting.
16. Incubate at room temperature for 2 min.
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Gwen Swinnen et al.