| Gene |
PCR |
Gene-specific Primer |
PCR Program |
|
| Ce11 |
5'RACE-1 |
cDNA: cDNA-as1: ATGAGAGCGAACAGCATGAC |
annealing: 50°C |
| canine |
|
inverse PCR: invCe11-s1: TATGGCATGGCTGCAGG |
extension: 1 min |
|
|
inveCe11-as1: GGCATCACGTCCACTG |
40 cycles |
|
5'RACE-2 |
cDNA: cDNA-as2 CCTGCACTTGGTTCCAACTG |
annealing: 55°C |
|
|
inverse PCR: invCe11-s2: TATGACCTGGCAGTAGTGGG |
extension: 1 min |
|
|
invCe11-as2: TGCTTGTCTGAGTGGTCAGG |
40 cycles |
|
5'RACE-3 |
cDNA: Ce11-as3: ACCTCCACGGGCCAGTCCAGCA |
annealing: TD 61-51°C |
|
|
inverse PCR: invCe11-s3: CTGCTGCTCAAGGTGCTGCT |
extension: 1 min |
|
|
invCe11-as3: CGTCAGGCCCACCACCAG |
40 cycles |
|
5'RACE-4 |
cDNA: Ce11-as4: CCACCAGGTTGTAGACGATGTAGAAGTTG |
annealing: TD 66-61°C |
|
|
inverse PCR: invCe11-s4: GCGCGGCCTCCAACCACAGC |
extension: 1 min |
|
|
invCe11-as4: CTTCACCACGAGGAGCAGCCCC |
40 cycles |
|
PCR-1 |
Ce11-s1: GTGCTAAGGGAGCCTCTCAG |
annealing: 58°C |
|
542 bp |
Ce11-as1: TTGCTGGTGTTCGCAGCTCC |
extension: 1 min |
|
Northern blot, in situ |
|
30 cycles |
|
PCR-2 |
Ce11-s2: GAGTCCCGGCCCGTCACC |
annealing: TD 65-55°C |
|
1984 bp |
Ce11-as2: GAGCCCGAGCCCCAAAATCTTC |
extension: 1 min |
|
full-length |
|
30 cycles |
|
PCR-3 |
Ce11-s3: GGAAAGCCAAGCCCCGGAGAACGAT |
annealing: TD 65-55°C |
|
620 bp |
Ce11-as3: GTCGCCAGCACCCAGTCCCTTCCTT |
extension: 2 min |
|
tissue screening |
|
25 cycles, 30 cycles |
| TSCOT |
PCR-1 |
HE11-s1: AAAACCCCATAAAACCACCATTG (exon1) |
annealing: TD 55-45°C |
| human |
937 bp |
HE11-as1: TCGCTGTAGCTGGAGTCTGAA (exon 4) |
extension: 1 min |
|
|
|
30 cycles |
|
PCR-2 |
HE11-s2: TGCTCTCATCCCCGTCACAAC (exon2) |
annealing: TD 55-45°C |
|
616 bp |
HE11-as1: TCGCTGTAGCTGGAGTCTGAA (exon4) |
extension: 1 min |
|
|
|
30 cycles |
|
PCR-3 |
HE11-s3: TCCTTCCTGGCCATCATTC (exon3) |
annealing: TD 55-45°C |
|
420 bp |
HE11-as1: TCGCTGTAGCTGGAGTCTGAA (exon4) |
extension: 1 min |
|
|
|
30 cycles |
|
PCR-4 |
HE11-s4: CATCGTGGCCTATAAACAAGTC (exon4) |
annealing: TD 55-45°C |
|
394 bp |
HE11-as1: TCGCTGTAGCTGGAGTCTGAA (exon4) |
extension: 1 min |
|
|
|
30 cycles |
| TSCOT |
PCR-1 |
Me11-s1: GGTGGCCGGCTCCCTCTACGAC (exon1) |
annealing: TD 69-61°C |
| mouse |
1323 bp |
Me11-as1: CCTTGTTGTGACCTCGGGACTTGTTTGTA (exon4) |
extension: 2 min |
|
|
|
30 cycles |
| Ce1 |
PCR-1 |
Ce1-s1: AAGCAGCAGAGCTGGAG |
annealing: 60°C |
| canine |
766 bp, control |
Ce1-as1: AATGTCACCTTCACCAG |
extension: 1 min |
|
Northern blot |
|
30 cycles |
|
| *Gene specific primer sequences (GenBank accessions: Ce11 AY195876, human
TSCOT AF242557, mouse TSCOT AF148145, Ce1 S77411). s and as indicate sense and
antisense; TD, touch down. |
