7. Set up a colony-PCR for four colonies using pGG-F and
pGG-R as primers and anneal at 55
C. The expected band
size is 610 bp.
8. Isolate binary vector DNA from two colonies using a plasmid
miniprep kit.
9. Conduct Sanger sequencing with pGG-R to verify the presence
of your spacer.
3.3.2 Golden Gate Entry
Module for Two gRNAs
1. For two gRNA target sites, order two oligonucleotides with
16-bp overhangs (see Notes 27, 28 and 33):
Forward: 5
0 -TTTTGAAGACAAATTG-protospacer (gRNA
target site 1)- GTTTTAGAGCTAGAAATAGC-3
0 ,
Reverse: 5
0 -TTTTGAAGACAAAAAC-reverse complement of
protospacer (gRNA target site 2)- CAATCACTACTTCGACTC-3
0 .
2. Set up a PCR using pEN-2xAtU6 as template and the oligonucleotides for the two gRNA target sites as primers and anneal
at 55
C. The expected band size is 580 bp.
3. Isolate the PCR amplicon using gel extraction (see Note 35).
4. Mix 1 μL of purified PCR product (~5 ng) with 1 μL of 50 ng/
μL entry vector (see Note 34), 5 units of BbsI-HF, 200 units of
T4 DNA ligase, 1.5 μL of 10 mM ATP, 1.5 μL of 10Â recommended restriction enzyme buffer and 9 μL of Milli-Q water in
a PCR tube.
5. Place in a thermal cycler and run the following program to
perform a Golden Gate reaction: (37
C for 3 min, 16
C for
3 min) Â 30 cycles, 50
C for 5 min, 80
C for 5 min, hold at
12
C.
6. Transform 5 μL of the Golden Gate reaction into competent E.
coli DH5α and plate on LB medium with 100 μg/mL
carbenicillin.
7. Set up a colony-PCR for four colonies using pGG-F and
pGG-R as primers and anneal at 55
C. The expected band
size is 1140 bp.
8. Isolate binary vector DNA from two colonies using a plasmid
miniprep kit.
9. Conduct Sanger sequencing with pGG-F and pGG-R to verify
the presence of your spacers.
3.3.3 Transfer
of gRNA(s) into
the Binary Vector
Here, six Golden Gate entry modules, with each of them containing either one gRNA, two gRNAs or a linker, are assembled into a
binary vector (see Note 2).
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Gwen Swinnen et al.