16. Transform ligation product into E. coli as described above
(Subheading 3.1.3, steps 7–9).
17. Screen correct clones by colony PCR using pCAMBIA1300specific P2 primer and reverse oligo for sgRNA (see Table 1).
Correct clones are expected to give PCR products of approximately 650 bp by following above procedure (Subheading
3.1.3, step 10).
18. Miniprep 2–4 clones using the QIAprep Spin Miniprep Kit and
confirm the AtU6-sgRNA cassette with Cas9 in pCAMBIA1300 by DNA sequencing using P2 primer (see Table 1).
3.1.4 Preparation of TDNA Construct for Multiple
Targets (Figs. 2 and 7)
1. Construct a certain number of sgRNA cassettes for multiple
target genes following the above procedure (see Subheading
3.1.3) (Figs. 3 and 6a).
2. If necessary, amplify some sgRNA cassettes to alter those
restriction enzyme sites by PCR using high-fidelity polymerase
(Fig. 3a, see Table 1):
AtU6–26-sgRNA forward with restriction enzyme site:
5
0 -restriction enzyme site-CATTCGGAGTTTTTGTATC
TTGTTTC-3’.
AtU6–26-sgRNA reverse with restriction enzyme site:
5
0 -restriction enzyme site-AAAAAAAGCACCGACTC
GGT-3
0 .
0.5 μL Template sgRNA Plasmid
PCR reaction buffer (final concentration 1Â).
1.6 μL dNTP (2.5 mM).
0.1 μL High-Fidelity Polymerase (e.g., KOD DNA
Polymerase)
0.4 μL AtU6–26-sgRNA forward with restriction enzyme site
primer (10 μM).
0.4 μL AtU6–26-sgRNA Reverse with Restriction Enzyme Site
Primer (10 μM)
Add ddH 2 O to 20 μL.
Perform PCR: 95
C for 5 min, 30 cycles of 94
C for 30 sec,
60
C for 30 s and 72
C for 1 min, and additional 5 min of
72
C.
3. Run a 1.2% agarose gel and gel-purify the PCR products by
using QIAquick Gel Extraction Kit. Correct clones will give
PCR products of approximately 550 bp. Elute the products
into 30 μL elution buffer. If necessary, subclone the PCR
products into Zero Blunt TOPO PCR Cloning according to
manufacturer’s instructions. Then, confirm the DNA sequence
(see Note 3).
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