2. Anneal each pair of oligomers in a thermocycler under the
following conditions: 95
C 5 min, and ramp down to 25
C
at 0.2
C/s.
3. Dilute annealed oligo 250 times with ddH 2 O.
4. Digest AtU6 sgRNA cassette plasmid with BbsI (Figs. 3a and
6a, b):
0.5 μg Plasmid.
1 μL BbsI (10 U/μL).
2 μL 10Â Reaction buffer.
Add ddH 2 O to 20 μL.
Incubate at least 1 h at 37
C.
Alternatively, AtU6 sgRNA cassette harboring binary vectors can be used as one plasmid system (Fig. 6b) [17]. If the
one plasmid system is applied, annealed oligo is directly cloned
into the binary vector. The use of restriction enzyme
(s) depends on the type of plasmid.
5. Run digested plasmid on a 0.8% TAE agarose gel and purify the
digested plasmid with QIAquick Gel Extraction Kit; elute the
digested plasmid into 30 μL elution buffer.
6. Ligate annealed oligo into BbsI-digested plasmid (Figs. 3a and
6a, b):
10 ng BbsI-digested plasmid.
1 μL Annealed oligo.
1 μL 10Â T4 DNA Ligase buffer.
0.5 μL T4 DNA Ligase.
Add ddH 2 O to 10 μL.
Incubate at 16
C overnight or room temperature for 10 min.
7. Transform 100 μL of E. coli DH5α chemically competent cells
with 5 μL of ligation product using a heat shock at 42
C for
45 s.
8. Recover transformed E. coli with 200 μL LB liquid medium
and incubate at 37
C for 1 h.
9. Spread 100 μL of transformed E. coli onto LB plate with
50 mg/L carbenicillin (or ampicillin in case of pBluescript SK
(Fig. 6a), otherwise suitable antibiotics should be applied).
Incubate at 37
C for overnight.
10. Screen for correct clones by colony PCR using primers
AtU6–26-sgRNA forward and reverse oligo for sgRNA (see
Table 1):
2 μL 10Â Taq buffer:
1.6 μL dNTP (2.5 mM)
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