Table 1. PCR primer sets.
Primers for amplification of: | Sequence | TA |
Unmodified vector | F: 5′-GGCCATTAATTTAATTAACCTTATTTTACAGATGG-3′ R: 5′-GGAAGGATCCACTAGTAGATTGGGAAAGCTGCTG-3′ | 59°C, 30 s |
Mut 1 vector | F: 5′-GTTCCTTTTGGTCCTAGGTCATTGTAAATCAG-3′ R: 5′-CTGATTTACAATGACCTAGGACCAAAAGGAAC-3′ | 60°C, 30 s |
Mut 7 vector | F: 5′-GGACTGGGGGTGGGGAGGGGATACAGGGTGTG-3′ R: 5′-CACACCCTGTATCCCCTCCCCACCCCCAGTCC-3′ | 60°C, 30 s |
Del vector | F: 5′-GGGAATTCCTCTCAAGGCTGGTGGGAG-3′ R: 5′-GGGAATTCGCTCTCTAACGTGATTA-3′ | 60°C. 30 s |
Oxtr mRNA | F: 5′-GTGCAGATGTGGAGCGTCT-3′ R: 5′-GTTGAGGCTGGCCAAGAG-3′ | 60°C, 30 s |
Hprt1 mRNA | F: 5′-GGGATTTGAATCACGTTTGTG-3′ R: 5′-TTGCGCTCATCTTAGGCTTT-3′ | 60°C, 30 s |
Bisulfite treated Oxtr First PCR round | 5′- TTGGTTTTTTTTGTTTTTTTTG-3′ 5′- AAAAAATATCTACCCCTCCCA-3′ | 54°C, 30 s |
Bisulfite treated Oxtr Second PCR round (Nested PCR) | 5′- TTGGTTTTTTTTGTTTTTTTTG-3′ 5′- CCACCAACCTTAAAAAAAATCT-3′ | 54°C, 30 s |
TA, Temperatures and time applied for annealing.