Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr19:4084768+4084882 115bp GGCCTTGGGTATGTTGTGAG CGATCCTGCAAAGTACACAGA
Mut= 109 bp
Wt= 115 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GTCCTGGGGTCCCATGGCAGGTCACAGGGAAAGGGATCCCTAAGACACCCAGACAGATGACCAGGACCGGGCAGAATCAGGCACAGGCCTTGGGTATGTTGTGAGGTGGGCTGGATGAGAAACCTCAAGACACAGAAAGAGGGTgccgaggtgtgggcaggccaaggaactcctaactatctgtgtactttgcaggatcgagagctgagaccagaagagatcgaaggtaatgggttcagggagtgggtagttgaggtggagtgtggggtgggggttacaagaagaggtttggggctagggcctctgcaaggtccaggaggagcgagaaggacatggggtctgggcagggagggctggcaaaacctcccttctttgccaaccccttggtctcagtgaccacactggtcaaggactgcttttatatttgtccatctggctgacacttcctggctgtccactgtgggcctggccccttgctaaccctgctaaccaggtagattaagggccccatcttctggagatagctacagctgcagctgcatagagcactcttaggaaaaccaatgactgggctgactaggaagaaatgggcagggaagtcttcctggtagcttttctgtctcttctcccctcccaccttctcttccttctctttttaatcacctagaacagagatgggcacatgacagatgcttcttaagcattggatgagagaacaaaattttaacactacacatgcaaaggctcttgggtaacaactaggaactgaggtttaacaacaagaagggactggagcaaagggaaaagatggaccggccccaaccggcagccaggaaggggcgctcagaaagggactaacctcactctgtccggcagagctgcagatcgccttccaggagtttgaccgagaccgggacggctacatcggctaccgggagctgggtgcctgcatgcggacactgggctacatgcccacggagatggagctcattgagatatcacaacagatcagtatgtgcttcagacccaacagagcctcatgccccagccctggcccagccctgaggtagacgtgtgcgctgttccaaacgcccaggaTATAAAGTTGTTATTAAAGGGAGAGCAGGGAGGATGGCTCCAGTAAAGAGACTGAAGCTCAGAGAGGTGAAGTATTGTCTAGAGTCACACAGCAGGGGCTCAGAGAAACCAAGAAACTGTGACTTAAGATCCTAGTA
This mutation is a 954 bp deletion beginning at Chromosome 19 position 4,084,827 bp and ending after 4,085,780 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
42297 | GGC CTT GGG TAT GTT GTG AG | Common | A | |||
42298 | CGA TCC TGC AAA GTA CAC AGA | Wild type Reverse | A | |||
42299 | CTC TTT ACT GGA GCC ATC CTC | Mutant Reverse | A | |||
42300 | Fluorophore-1 | CAG GCC AAG GAA CTC CTA ACT | Quencher-1 | WT Probe | ||
42301 | Fluorophore-2 | AGA GGG TTA TAA AGT TGT TAT TAA AGG GA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
42297 | 0.40 uM |
42298 | 0.40 uM |
42299 | 0.40 uM |
Wt Probe | 0.15 uM |
Mutant Probe | 0.15 uM |
DNA |
Step | Temp °C | Time | Note |
---|---|---|---|
1 | 95.0 | -- | |
2 | 95.0 | -- | |
3 | 60.0 | -- | |
4 | -- | repeat steps 2-3 for 40 cycles | |
5 | 40.0 | -- | Forever |