12. Add 300 μL isopropanol to each tube, invert tubes, and
mix well.
13. Centrifuge at maximum speed for 10 min, and discard
supernatant.
14. Add 500 μL of 75% ethanol.
15. Centrifuge at maximum speed for 5 min, and discard
supernatant.
16. Centrifuge those tubes again to spin down all aliquot.
17. Remove all aliquots by using pipet.
18. Add 20–50 μL ddH 2 O to solve the DNA.
19. Design PCR primers that amplify the on-target site (Figs. 3b
and 8a). If you used a sgRNA design website such as CRISPOR
(see Subheading 3.1.1), the website also designs genotyping
primers and certain restriction enzyme sites.
20. Perform PCR by including a wild-type (WT) DNA sample
(e.g., Col-0) as a control:
0.5 μL Template extracted genome DNA (from Subheading
3.1.6, step 18).
2 μL 10Â Taq buffer.
1.6 μL dNTP (2.5 mM).
0.05 μL Taq polymerase.
0.4 μL Forward primer (10 μM).
0.4 μL Reverse primer (10 μM)
Add ddH 2 O to 20 μL.
Perform PCR: 95
C for 5 min, 35 cycles of 94
C for 30 s,
60
C for 30 s and 72
C for 1 min, and additional 5 min of
72
C.
21. Digest the PCR products (Fig. 8a):
10 μL PCR product.
2 μL 10Â Reaction buffer.
0.5 μL certain restriction enzyme (e.g. PstI, Fig. 3b)
Add ddH 2 O to 20 μL.
Incubate at 37
C at least 1 h.
22. Run digested products on a 1.5% agarose gel. PCR product
form control WT, such as Col-0, must be completely digested
while products from modified samples should be undigested
band (Fig. 8a, b).
23. Purify the undigested band by using QIAquick Gel Extraction
Kit, and identify the mutation by sequencing (Fig. 8b, c).
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