1.6 μL dNTP (2.5 mM).
0.1 μL High-fidelity polymerase (e.g., KOD DNA
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, 30 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.
4. Run PCR products on a 0.8% agarose gel and gel-purify them
by using QIAquick Gel Extraction Kit; elute the PCR products
into 30 μL elution buffer. If necessary, subclone the PCR
products into Zero Blunt TOPO PCR Cloning according to
manufacturer’s instruction, then confirm DNA sequences (see
Note 3).
5. Digest AtU6-sgRNA cassette, donor DNA, and pCAMBIA3301 binary vector with certain restriction enzymes:
10 μL AtU6-sgRNA in pBluescript SK (see Subheading 3.2.1).
1 μL HindIII.
1 μL SalI.
5 μL CutSmart Buffer.
Add ddH 2 O to 50 μL.
10 μL donor DNA PCR product or subcloning in Zero Blunt
TOPO (see Subheading 3.2.3, step 4).
1 μL EcoRI.
1 μL SalI.
5 μL CutSmart Buffer.
Add ddH 2 O to 50 μL.
10 μL pCAMBIA3301.
1 μL HindIII.
1 μL EcoRI.
5 μL CutSmart Buffer.
Add ddH 2 O to 50 μL.
Incubate reactions at 37
C for 1 h to overnight.
6. Run digested plasmids on a 1.2% agarose gel. Gel-purify the
insert AtU6-sgRNA, donor DNA, and linearized pCMABIA3301 by using QIAquick Gel Extraction Kit and elute the
digested plasmid into 30 μL elution buffer.
7. Determine the concentration of digested inserts and vector by
spectrometer.
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