Table 3 PCR primers used in this study
From: Efficient generation of Rosa26 knock-in mice using CRISPR/Cas9 in C57BL/6 zygotes
PCR type | Primer | Sequence (5′ > 3′) |
Nested PCR for Rosa26 locus | R26F1 | CCAAAGTCGCTCTGAGTTGTTATCAGT |
R26R1 | GGAGCGGGAGAAATGGATATGAAG | |
R26F2 | GCCTCCTGGCTTCTGAGGACCG | |
R26R2 | TCTGTGGGAAGTCTTGTCCCTCC | |
Cas9 transgene | Cas9F | GGCATCCTGCAGACAGTGAAGGTGG |
Cas9R | CGGTTCTTGTCGCTTCTGGTCAGCA | |
Homozygous | ||
Heterozygous for R26-Cas9 allele | R26F2 | GCCTCCTGGCTTCTGAGGACCG |
R26R2 | TCTGTGGGAAGTCTTGTCCCTCC | |
SAR | CCTGGACTACTGCGCCCTACAGA | |
Long PCR for correct | ||
Integration | R26F3 | CTGCCCGAGCGGAAACGCCACTGAC |
SAR | CCTGGACTACTGCGCCCTACAGA | |
Neo transgene | NeoF | GCTAACCATGTTCATGCCTTC |
NeoR | CGTTGGCTACCCGTGATATT | |
Loading PCR for Rosa26 locus | R26wtF | GGAGTGTTGCAATACCTTTCTGGGAGTTC |
R26wtR | TGTCCCTCCAATTTTACACCTGTTCAATTC | |
Lgals-Cd274 transgene | Lgals1F | CAAGATTAAGTGCGTGGCC |
Cd274R | CATTTCCCTTCAAAAGCTGGTC | |
Venus correct integration | VenusF | GGCCTCTCGAGCCTCTAGAACTATAGTG |
R26R3 | CAAGCTCACAAGACCTTAGGTCAGGA | |
LoxP-flanked Stop cassette deletion | CagF | CAAGCTCACAAGACCTTAGGTCAGGA |
NeoR1 | ATGGGATCGGCCATTGAACAAGATGG | |
Cas9R1 | CGGCCTTGTCGGTGCTGTCCACCAG | |
PCR Rosa26 for T7EI and RLFP | R26T7F | CGTGCAAGTTGAGTCCATCCGCC |
R26T7R | ACTCCGAGGCGGATCACAAGCA |