22. If the protospacer were to contain a BpiI or BsaI site, this
would interfere with the MultiSite-Puchta vector construction
in Subheading 3.2 and the modular vector construction in
Subheading 3.3.
23. The Cas-cleavage site should be located 150–250 bp from the
forward or reverse primer to enable downstream analysis using
ICE in Subheading 3.5.3.
24. One PCR primer pair might be used to amplify two gRNA
target sites if they are located near each other, with either
Cas-cleavage site located 150–250 bp from each primer.
25. Use Primer-BLAST to ensure specific amplification of your
target region.
26. This step should be performed prior to any vector construction. A reliable PCR amplicon is required to simplify downstream analysis. Possible problems with amplification and
sequencing can be (a) that the material in your hands does
not match the reference sequence, (b) the presence of highly
homologous sequences, resulting in a mixture of PCR products and (c) the presence of homopolymer region(s) within
the amplicon, which will result in overlapping chromatograms.
27. The PAM sequence is not included in the oligonucleotides.
28. The 5
0 overhang of the forward oligonucleotide contains the G
initiation nucleotide for the AtU6–26 polymerase III
promoter.
29. The overhangs are complementary with the sticky ends generated by BpiI in the chosen entry vector in steps 4–5 resulting in
seamless cloning.
30. Use pENC1.1 to transfer one gRNA to a binary vector in
Subheading 3.2.2. To transfer two gRNAs to a binary vector
in Subheading 3.2.2, use pMR217 for your first gRNA and
pMR218 for your second (see Table 2).
31. Use LB with 100 μg/mL carbenicillin for a cut-ligation reaction with pENC1.1 and LB with 75 μg/mL kanamycin for a
cut-ligation reaction with pMR217 or pMR218.
32. Use a binary vector according to your preference (see Table 2).
33. The overhangs are complementary with the sticky ends generated by BbsI-HF in the chosen entry vector in steps 4–5
resulting in seamless cloning.
34. Use an entry vector according to your preference (see Table 3).
35. In our experience, gel extraction of the PCR amplicon yields
better results than PCR purification.
36. Use a binary vector according to your preference (see Table 3).
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Gwen Swinnen et al.
would interfere with the MultiSite-Puchta vector construction
in Subheading 3.2 and the modular vector construction in
Subheading 3.3.
23. The Cas-cleavage site should be located 150–250 bp from the
forward or reverse primer to enable downstream analysis using
ICE in Subheading 3.5.3.
24. One PCR primer pair might be used to amplify two gRNA
target sites if they are located near each other, with either
Cas-cleavage site located 150–250 bp from each primer.
25. Use Primer-BLAST to ensure specific amplification of your
target region.
26. This step should be performed prior to any vector construction. A reliable PCR amplicon is required to simplify downstream analysis. Possible problems with amplification and
sequencing can be (a) that the material in your hands does
not match the reference sequence, (b) the presence of highly
homologous sequences, resulting in a mixture of PCR products and (c) the presence of homopolymer region(s) within
the amplicon, which will result in overlapping chromatograms.
27. The PAM sequence is not included in the oligonucleotides.
28. The 5
0 overhang of the forward oligonucleotide contains the G
initiation nucleotide for the AtU6–26 polymerase III
promoter.
29. The overhangs are complementary with the sticky ends generated by BpiI in the chosen entry vector in steps 4–5 resulting in
seamless cloning.
30. Use pENC1.1 to transfer one gRNA to a binary vector in
Subheading 3.2.2. To transfer two gRNAs to a binary vector
in Subheading 3.2.2, use pMR217 for your first gRNA and
pMR218 for your second (see Table 2).
31. Use LB with 100 μg/mL carbenicillin for a cut-ligation reaction with pENC1.1 and LB with 75 μg/mL kanamycin for a
cut-ligation reaction with pMR217 or pMR218.
32. Use a binary vector according to your preference (see Table 2).
33. The overhangs are complementary with the sticky ends generated by BbsI-HF in the chosen entry vector in steps 4–5
resulting in seamless cloning.
34. Use an entry vector according to your preference (see Table 3).
35. In our experience, gel extraction of the PCR amplicon yields
better results than PCR purification.
36. Use a binary vector according to your preference (see Table 3).
338
Gwen Swinnen et al.