PCR Reactions to Amplify Each Exon of CACNA1S Using the Following Reagents and Conditions

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