PAX6, CHX10, and SIX3 primers and PCR conditions
| Forward oligonucleotide | Reverse oligonucleotide | PCR conditions | |
|---|---|---|---|
| *Love et al. Hum Mutat 2000;12:128-34. | |||
| PCR reagents were essentially as described in Love et al, except the polymerase used was AmplitaqGold (Amersham) supplemented with cloned Pfu (Stratagene). | |||
| PAX6 | |||
| Exons 1–13 | As in Love et al* | As in Love et al* except for exon 13 (5′-TTTTACTGTAATCTTGGCCAG) | Exons 1–5a touchdown with 10% DMSO: 95°C, 2 min [94°C, 1 min; 55–48°C, 1 min; 72°C, 1 min]×40; 72°C, 10 min Exons 6–13: 95°C, 2 min [94°C, 1 min; 53–61°C, 1 min; 72°C, 1 min]×40; 72°C, 10 min |
| SIX3 | |||
| 5′UTR/CDS | 5′-AAATGTTTCAATGCTGCACTC | 5′-GGAGCGGTGGTGAGAATC | 10% DMSO: 94°C, 4 min [98°C, 45 sec; 53°C, 1 min; 72°C, 3 min]×40; 72°C, 5 min |
| SIX domain | 5′-CTAGACCTCTATTCCTCCC | 5′-CTCAAGGATGTGGTAGAGG | 10% DMSO: 95°C, 2 min [94°C, 1 min; 53°C, 1 min; 72°C, 1 min]×40; 72°C, 10 min |
| SIX/homeodom | 5′-GTGGTCGCCTTCCACACG | 5′-CTTCCCAGGCCTCTGAGGC | 10% DMSO: 95°C, 2 min [94°C, 1 min; 55°C, 1 min; 72°C, 1 min]×40; 72°C, 10 min |
| CDS/3′UTR | 5′-GTGGCGGGCCTCTGTGTC | 5′-TTTGGCTGGGGTGGGAGTG | 10% DMSO: 94°C, 4 min [98°C, 45 sec; 57°C, 1 min; 72°C, 3 min]×40; 72°C, 5 min |
| 3′UTR | 5′-CGAAATCAGGATACCCAACC | 5′-CGGAACGGAGACGGCGAC | 10% DMSO: 95°C, 2 min [94°C, 1 min; 54°C, 1 min; 72°C, 1 min]×40; 72°C, 10 min |
| CHX10 | |||
| Exons 1–5 | As in ref 6 | As in ref 6 | Exon 1 touchdown with 10% DMSO: 95°C, 2 min [94°C, 1 min; 67–63°C, 1 min; 72°C, 1 min]×40; 72°C, 10 min |
| Exons 2–5: 95°C, 2 min [94°C, 1 min; 63–65°C, 1 min; 72°C, 1 min]×40; 72°C, 10 min | |||