PCR Conditions
PCR reactions to amplify each exon of CACNA1S using the following reagents and conditions were performed: A 25μL reaction contained 200ng genomic DNA, 5μL of 10 x PCR buffer without MgCl2 (Applied Biosystems), 4μL of 25mMMgCl2, 5μL of 2mMdNTPs, 15pmol of each primer (forward and reverse), and 2.5 units of AmpliTaq Gold polymerase (Applied Biosystems). PCR reactions to amplify each exon of SCN4A using the following reagents and conditions were additionally performed. A 50μL reaction contained 200ng genomic DNA, 10μL of 5 x PCR buffer(Promega), 5μL of 2mMdNTPs, 10pmol of each primer (forward and reverse), and 1.0 unit of Go-Taq polymerase (Promega).
Cycling conditions consisted of an initial denaturing step of 95 °C for 10 minutes followed by 30 cycles of 95 °C for 30 seconds, 60 °C for 30 seconds, 72 °C for 30 seconds, and a final extension step of 72 °C for 7 minutes. Samples were sequenced (bidirectionally) using the ABI Big Dye Terminator Sequencing Kit version 1.1, M13 universal primers, and an ABI Model 3730xl Automated DNA Sequencer. Data were analyzed using version 2.5 SeqScape Analysis software (ABI).
Primer Sequences
All primers are tagged with M13 universal primers.
Exon 4 CACNA1S
4F TGTAAAACGACGGCCAGTTATTTGCCGTCTCTCCCCTA
4R CAGGAAACAGCTATGACCTAGGAAGGGGACCCAGAACT
Exon 11 CACNA1S
11F TGTAAAACGACGGCCAGTGGGAGTCAGGAGAAGGGAAG
11R CAGGAAACAGCTATGACCAACCTGCACAGATCCCAGAC
Exon 21 CACNA1S
21F TGTAAAACGACGGCCAGTGGAAGGTGGGAGTGTTTTCA
21R CAGGAAACAGCTATGACCAGACTTGCTGCCTCCTGATG
Exon 30 CACNA1S
30F TGTAAAACGACGGCCAGTAGTTCCTCCATGCCACAAAC
30R CAGGAAACAGCTATGACCGAGCGCCAGTCAGTGTCTTA
Exon 5 SCN4A
5F TGTAAAACGACGGCCAGTAGGAGCTTTGGGGGTGTCT
5R CAGGAAACAGCTATGACCATCTGCCCTCTGGTCACG
Exon 12 SCN4A
12 F TGTAAAACGACGGCCAGTGGAGTTGGGTGGGAGACTTC
12R CAGGAAACAGCTATGACCTTCAGCCCTCTAGCTTCCTG
Exon 13 SCN4A
13F TGTAAAACGACGGCCAGTCTGCCTTGGGTGGTGGTC
13R CAGGAAACAGCTATGACCTTGGGGAGATAGAAAACAGTCA
Exon 18 SCN4A
18F TGTAAAACGACGGCCAGTGTGCCAGGCTCCTACAGGT
18R CAGGAAACAGCTATGACCCTCTCTGCTTCCCTCTGGTG
Exon 24 SCN4A
24F: TGTAAAACGACGGCCAGTCTCTGAGACTTGAGCAGAGCAC
24R: CAGGAAACAGCTATGACCTCTCGAACAGGCAGATGATG