SSCP CONDITIONS for IRBP (RBP3):
|
Exon |
Primer |
Primer sequence |
Temp |
[MgCl2] |
pH |
DMSO |
Enzyme Digest |
Size (bp) |
|
1A |
1256 |
GCTTGCACACAGTCCAGGGA |
58 |
1.0 |
8.4 |
10% |
MspI |
103,127,180(409) |
|
1A |
1257 |
TTGCAGCCAGGCAAGCAGTT |
||||||
|
1B |
1258 |
ACAAGTCCCAGCACTCACCA |
56 |
1.0 |
8.4 |
10% |
MspI |
92,115,198 (405) |
|
1B |
1259 |
GGTCTGGCTGCTGGTGAGGA |
||||||
|
1C |
1260 |
GAAAGGTACGGTGCCGACAA |
56 |
1.0 |
8.4 |
10% |
MspI |
102,125,143,200(569) |
|
1C |
1261 |
TGAGTCTTCGGCTGCAGCGT |
||||||
|
1D |
1262 |
CCACAGAAACTCCTTCTTGG |
56 |
1.5 |
8.6 |
10% |
Taq1 |
157,198(355) |
|
1D |
1263 |
ATAGGTGGTGAAGAGGTGCA |
||||||
|
1E |
1264 |
TCCTACTTCCAGGGCCCTGA |
54 |
1.0 |
8.6 |
10% |
MspI |
77,127,201(427) |
|
1E |
1265 |
AGGCTTTGGTGGAACTCCAG |
||||||
|
1F |
1266 |
TGGACAAAGCCCAGGAAGTG |
56 |
1.0 |
8.6 |
10% |
PvuII |
63,156,200(437) |
|
1F |
1267 |
ATACCAGCCGCACCAGCTGT |
||||||
|
1G |
1268 |
CTGAACTGGAGACAGTGAAG |
52 |
1.0 |
8.6 |
10% |
BstNI |
100,127,165(404) |
|
1G |
1269 |
TGCTGCCCACCTGGTAGATG |
||||||
|
1H |
1270 |
GGAGGCGCACTCTCTGTGGG |
54 |
1.0 |
8.6 |
10% |
StyI |
70,183,216 (470) |
|
1H |
1271 |
GGGACTGGGTTCAGCCAGGT |
||||||
|
2 |
1272 |
CCTCCTGACACTGAGTAGGA |
52 |
1.0 |
8.4 |
10% |
AluI |
61,80,120(242) |
|
2 |
1273 |
TCCTGGCTAGGAAGCACTGA |
||||||
|
3 |
1274 |
GAACAGGCTCTGCTTCCCAT |
52 |
1.5 |
8.4 |
10% |
None |
194 |
|
3 |
1275 |
GAAAGGAGAAAGCTTCTCCA |
||||||
|
4 |
1276 |
CCATCCTGAAGGGCCTTATG |
54 |
1.0 |
8.6 |
10% |
EcoO109 |
41,107,118,153(432) |
|
4 |
1277 |
TGTCCCAGAGGTTCTGTCTG |
Amplification Conditions for IRBP for sequencing(RBP3):
(Used internal primers to sequence exon 1, see below. Also, used 10X amp buffer from Applied Biosystems containing no MgCl2). Polymerization time for exon 1 was extended to 3 minutes because of size of fragment (35 cycles). 5M Betaine was substituted for DMSO.
|
Exon |
Primer |
Primer Sequence |
Temp |
[MgCl2] |
ul 5M Betaine /20ul rxn |
Size (bp) |
|
1 |
3236 |
TGCAATGTACCTTCTGGGTC |
58 |
2.0 |
7ul |
3344 |
|
1 |
1271 |
GGGACTGGGTTCAGCCAGGT |
58 |
2.0 |
7ul |
|
|
2 |
1272 |
CCTCCTGACACTGAGTAGGA |
54 |
2.0 |
7ul |
242 |
|
2 |
1273 |
TCCTGGCTAGGAAGCACTGA |
54 |
2.0 |
7ul |
|
|
3 |
1274 |
GAACAGGCTCTGCTTCCCAT |
54 |
2.0 |
7ul |
194 |
|
3 |
1276 |
GAAAGGAGAAAGCTTCTCCA |
54 |
2.0 |
7ul |
|
|
4 |
1276 |
CCATCCTGAAGGGCCTTATG |
54 |
2.0 |
7ul |
432 |
|
4 |
1277 |
TGTCCCAGAGGTTCTGTCTG |
54 |
2.0 |
7ul |
For sequencing of exon 1, the above internal primers were used (see table for SSCP conditions) to cover the exon: 1257, 1258, 1259, 1260, 1261, 1262, 1263, 1264, 1265, 1266, 1267, 1268, 1269, and 1270. Also another forward internal primer, 1955 (CTGCACAGAGCAGGGCCACG) was used for exon 1 (fragment 1A).
Cold (non-radioactive) Amplification Conditions for RDH5
|
Exon |
Primer |
Primer sequence |
Temp |
[MgCl2] |
pH |
DMSO |
Size (bp) |
|
1 |
4677 |
TGGGAGCAGTCTCTTAAACAAA |
58 |
1.5 |
8.4 |
10% |
299 |
|
1 |
4678 |
CCAAGAGGTTTGCTGTGTGA |
|||||
|
2A |
2719 |
GGCCACAGTAAACTGGACAA |
56 |
1.5 |
8.4 |
no |
178 |
|
2A |
3731 |
AGCCGGTGATGAAGACAAAG |
|||||
|
2B |
3732 |
TTACTCTGGGCAGTGCTGTG |
60 |
1.0 |
8.6 |
10% |
140 |
|
2B |
3733 |
AGGACTCGGAAGCCTCTCTG |
|||||
|
2C |
3734 |
TTCTGGCACTGCAGCTGGAC |
50 |
0.25 |
8.6 |
10% |
207 |
|
2C |
2718 |
TTCCTGGTGGTCTACCATAC |
|||||
|
3A |
2722 |
CCCCAGCATCCTTTTCATCT |
54 |
0.5 |
8.6 |
no |
210 |
|
3A |
3735 |
GACGCTGGTGATGTTGATCA |
|||||
|
3B |
3736 |
TGAACACAATGGGTCCCATC |
50 |
0.5 |
8.4 |
10% |
202 |
|
3B |
2723 |
TGTTAGTCCTGGAACCCAGG |
|||||
|
4 |
2724 |
AAGAACCCAGCAACTTCGCT |
58 |
1.0 |
8.4 |
10% |
237 |
|
4 |
2725 |
TTCCCTTCATGTGCCCCTGT |
|||||
|
5A |
3737 |
CTGATTGCAACCACCTATGG |
58 |
1.0 |
8.6 |
10% |
166 |
|
5A |
3738 |
AGAGCAGCTTGGCATCCCAA |
|||||
|
5B |
3739 |
TAACCAAGGTGAGCCGATGC |
58 |
1.5 |
8.4 |
no |
200 |
|
5B |
3740 |
CAATCTCTTGCTGGAAGGCT |
SSCP conditions for RDH5
|
Exon |
Primer |
Primer sequence |
Temp |
[MgCl2] |
pH |
DMSO |
Enzyme Digest |
Size (bp) |
|
1 |
4677 |
TGGGAGCAGTCTCTTAAACAAA |
58 |
1.5 |
8.4 |
10% |
299 |
|
|
1 |
4678 |
CCAAGAGGTTTGCTGTGTGA |
||||||
|
2A |
2719 |
GGCCACAGTAAACTGGACAA |
56 |
0.5 |
8.4 |
10% |
none |
178 |
|
2A |
3731 |
AGCCGGTGATGAAGACAAAG |
||||||
|
2B |
3732 |
TTACTCTGGGCAGTGCTGTG |
60 |
1.0 |
8.4 |
10% |
none |
140 |
|
2B |
3733 |
AGGACTCGGAAGCCTCTCTG |
||||||
|
2C |
3734 |
TTCTGGCACTGCAGCTGGAC |
50 |
.25 |
8.6 |
10% |
none |
207 |
|
2C |
2718 |
TTCCTGGTGGTCTACCATAC |
||||||
|
3A |
2722 |
CCCCAGCATCCTTTTCATCT |
54 |
1.0 |
8.4 |
10% |
none |
210 |
|
3A |
3735 |
GACGCTGGTGATGTTGATCA |
||||||
|
3B |
3736 |
TGAACACAATGGGTCCCATC |
50 |
0.5 |
8.4 |
10% |
none |
202 |
|
3B |
2723 |
TGTTAGTCCTGGAACCCAGG |
||||||
|
4 |
2724 |
AAGAACCCAGCAACTTCGCT |
58 |
1.5 |
8.4 |
no |
none |
237 |
|
4 |
2725 |
TTCCCTTCATGTGCCCCTGT |
||||||
|
5A |
3737 |
CTGATTGCAACCACCTATGG |
58 |
1.0 |
8.4 |
no |
none |
166 |
|
5A |
3738 |
AGAGCAGCTTGGCATCCCAA |
||||||
|
5B |
3739 |
TAACCAAGGTGAGCCGATGC |
58 |
1.5 |
8.4 |
10% |
none |
200 |
|
5B |
3740 |
CAATCTCTTGCTGGAAGGCT |
Amplification Conditions for CRALBP (RLBP1):
|
Exon |
Primer |
Primer sequence |
Temp |
[MgCl2] |
pH |
DMSO |
Size (bp) |
|
1 |
4673 |
gggcagagaaaagagggaat |
60 |
1.5 |
8.6 |
no |
432 |
|
1 |
4674 |
atcttgggacgtggtctcc |
|||||
|
2 |
4675 |
gcaggagagaaaacctggaa |
60 |
1.5 |
8.6 |
no |
352 |
|
2 |
4676 |
aggaaacgggaggaggaata |
|||||
|
3 |
3532 |
TGAGATCCACAGTTCTGAGAC |
58 |
1.0 |
8.6 |
no |
|
|
3 |
3533 |
AGGAGAGCCCTGGAGGACA |
|||||
|
4 |
3534 |
GGCTGATGCGGTTGGCTGTT |
58 |
0.5 |
8.6 |
10% |
|
|
4 |
3535 |
CCCCTCATGTTGCCTCCCTA |
|||||
|
5 |
3536 |
CTCATCACCTGTGTGTCCTGCC |
58 |
0.5 |
8.4 |
10% |
|
|
5 |
3537 |
GAGAGCGGATAGCATCCTCATG |
|||||
|
6 |
3538 |
CTTCTGAGTCCCACTAGGAGG |
58 |
1.0 |
8.4 |
no |
|
|
6 |
3539 |
CCAGTAGAGGCCAGGGTTGA |
|||||
|
7 |
3540 |
CCTCAGGACCTCAAGCCTTA |
58 |
0.5 |
8.4 |
10% |
|
|
7 |
3541 |
CTGCAAGCACCATGAAAGGA |
|||||
|
8 |
3542 |
AATGAGTGGGAGCCTCTGAG |
58 |
1.0 |
8.6 |
10% |
|
|
8 |
3543 |
CCCTCTTGTCTCATTGTCTGG |
|||||
|
9 |
3544 |
CTCCTGCTCAGTTCTGTCTC |
58 |
0.5 |
8.6 |
no |
|
|
9 |
3545 |
AGTTCAGCTGGCAGGAGATG |
Amplification Conditions for RDH8:
|
Exon |
Primer |
Primer sequence |
Temp |
[MgCl2] |
pH |
DMSO |
Enzyme Digest? |
Size (bp) |
|
1F |
4598 |
atctgtgggagggggttag |
54 |
2.0 |
8.6 |
- |
- |
240 |
|
1R |
4599 |
acccggctgcctcctagtg |
||||||
|
2F |
4600 |
aaaggagctcagtgcctgtc |
54 |
1.5 |
8.6 |
10% |
- |
247 |
|
2R |
4601 |
agaccaggatggggcttagt |
||||||
|
3F |
4602 |
gagggactttaaggtaaccc |
52 |
1.5 |
8.6 |
10% |
- |
250 |
|
3R |
4603 |
ggatttccagggttgggt |
||||||
|
4F |
4604 |
caccctggaacccacaaag |
54 |
2.0 |
8.6 |
- |
- |
207 |
|
4R |
4605 |
ccccatcactggctccaag |
||||||
|
5F |
4606 |
tggggcatcagacttacact |
54 |
1.5 |
8.6 |
10% |
- |
254 |
|
5R |
4607 |
cacgtcttggggctctgg |
||||||
|
6F |
4608 |
gacgtggctcaggtatgttg |
54 |
1.5 |
8.6 |
10% |
EarI |
328 |
|
6R |
4609 |
ggtctggttgttcaggatgg |