Add ddH 2 O to 20 μL.
Perform PCR: 95
C for 5 min, 35 cycles of 94
C for 30 sec,
55
C for 30 sec, and 72
C for 1 min, and additional 5 min
to 72
C.
Correct clones will give PCR products of approximately
470 bp.
11. Miniprep 2–4 clones using the QIAprep Spin Miniprep Kit and
confirm the AtU6-sgRNA cassette in pBluescript SK by DNA
sequencing.
12. Digest AtU6-sgRNA cassette and Cas9 harboring pCAMBIA1300 binary vector with restriction enzymes (Fig. 6a):
10 μL AtU6-sgRNA in pBluescript SK.
1 μL HindIII.
1 μL SalI.
5 μL CutSmart Buffer.
Add ddH 2 O to 50 μL.
10 μL Cas9 harboring pCAMBIA1300.
1 μL HindIII.
1 μL SalI.
5 μL CutSmart Buffer.
Add ddH 2 O to 50 μL.
Incubate at 37
C for 1 h to overnight.
13. Run digested plasmids on a 1.2% agarose gel. Gel-purify the
insert AtU6-sgRNA and linearized Cas9 harboring pCMABIA1300 by using QIAquick Gel Extraction Kit and elute the
digested plasmid into 30 μL elution buffer.
14. Determine the concentration of digested insert and vector by
spectrometer.
15. Clone AtU6-sgRNA cassette into the Cas9 harboring pCAMBIA1300 (Fig. 6a):
X μL AtU6-sgRNA cassette (insert).
10 ng Cas9 harboring pCAMBIA1300.
1 μL 10Â T4 DNA Ligase buffer.
0.5 μL T4 DNA Ligase
Add ddH 2 O to 10 μL.
The ratio of insert DNA mass (insert:vector ¼ 3 ~ 5:1) can be
calculated using a website (https://nebiocalculator.neb.
com/#!/ligation). Incubate at 16
C overnight or room
temperature for 10 min.
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