Region/gene | Primer, forward | Primer, reverse | PCR conditions |
---|---|---|---|
Xist, region 1A – 1st round | MM3, gttaattaatgtagaagaatttttagtgttta | MM2, aaatattcccccaaaactccttaaataa | 94°C for 15 minutes; (94°C for 20 seconds; 54°C for 30 seconds; 72°C for 90 seconds) × 25 cycles; 72°C for 5 minutes |
Xist, region 1A – nested | Tag 6F, aggaagagag ttgtaaaataggataattttttattat | T7 6R, cagtaatacgactcactatagggagaaggct atattcccccaaaactccttaaata | 94°C for 15 minutes; (94°C for 20 seconds; 54°C for 30 seconds; 72°C for 30 seconds) × 5 cycles; (94°C for 20 seconds; 60°C for 30 seconds; 72°C for 30 seconds) × 25 cycles; 72°C for 5 minutes |
Xist, region 2C | Tag 1F, aggaagagag attttgtttttaaattgagtgggtg | T7 1R, cagtaatacgactcactatagggagaaggct taaaccctatcccctaatcctctac | 94°C for 15 minutes; (94°C for 20 seconds; 60°C for 30 seconds; 72°C for 1 minute) × 5 cycles; (94°C for 20 seconds; 68°C for 30 minutes; 72°C for 1 minute) × 40 cycles; 72°C for 5 minutes |
Xist, region 2D | Tag 5F, aggaagagag agggtgtgtgtgtatatggattttt | T7 MM5, cagtaatacgactcactatagggagaaggct taactcaaacataaaactaacatataacaca | 94°C for 15 minutes; (94°C for 20 seconds; 54°C for 30 seconds; 72°C for 1 minute) × 5 cycles; (94°C for 20 seconds; 58°C for 30 seconds; 72°C for 1 minute) × 40 cycles; 72°C for 5 minutes |
Igf2r | Tag Igf2r_F2, aggaagagag gtgtggtatttttatgtatagttagg | T7 Igf2r_R cagtaatacgactcactatagggagaaggct aaatatcctaaaaatacaaactacac | 94°C for 15 minutes; (94°C for 20 seconds; 54°C for 30 seconds; 72°C for 1 minute) × 45 cycles; 72°C for 5 minutes |
H19A | Tag H19A, aggaagagag gagaaaatagttattgtttatagtttt | T7 H19A, cagtaatacgactcactatagggagaaggct cctaaaatactaaacttaaataacccacaa | 94°C for 15 minutes; (94°C for 20 seconds; 50°C for 30 seconds; 72°C for 1 minute) × 45 cycles; 72°C for 5 minutes |
H19B | Tag H19A, aggaagagag ttagtgtggtttattataggaaggtatagaagt | T7 H19A, cagtaatacgactcactatagggagaaggct taaacctaaaatactcaaactttatcacaac | 94°C for 15 minutes; (94°C for 20 seconds; 50°C for 30 seconds; 72°C for 1 minute) × 5 cycles; (94°C for 20 seconds; 58°C for 30 seconds; 72°C for 1 minute) × 40 cycles; 72°C for 5 minutes |