Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr13:67593824-67593920 97bp CACAGAATGCAGAGAATATGGA TTCACTCCTGTAGGGCTTCAC
Mut= 106 bp
Wt= 97 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GGCTTCATTGAATACAGGTGTAAAAAAATGAATGTAGCTTATCATTTATAAAGACAAGTGCAAGTAAGACAAGACAAGTGAGAAATGTTGGTTTTTAAAGAGAAACATTATAAATTTACAGAATTTTACTCTGCTTTTATTAATTTCTCAATCGTTTCTCCACAGAATGCAGAGAATATGGAAaagtttttgagtggtccaaagtgtttcaaaatcaccagataattgacttagaagtgaagccctacaggagtgaagaacgtgccaagtccttccatattccatcattaagttctgaagagagcacaattcatgcaggagaaaaaccttacaaatgtgaagtttgtggcaaggccttctgcattccattattactttctaaacacaagataattcataaaggagagaatctctacaagtgtgaagtgtgtggcaaggccttccaacatccatcaagactttccagacataagaagattcattcagaagagaaaccatacaagtgtgaagtatgtggaaaagccttccactttccatcattacttttggtacacaagagagttcatacaggagaaaaaccatacaagtgtgaagtatgtggcaaggcctttcattatccatcaatactctctaaacacaagcgaattcatacaggagagaaaccatacaagtgtgaagaatgtggcaaggccttccacatttcatcatttctttctaaacacaaaataattcatagaggagaaaaaccctacaagtgtgaggtatgtggcaaggccttccattatccatcaagactttccaaccataagaaaatccattcagtagagaaaccatacaagtgtgaaatatgtgggaaggccttccgcattttatcactcctttctaagcacaagataattcatacagaagagaatccttacaagtgtgaagtatgtggcaaggctttcgattatccctcaagactttctacccatgcaaaaatgcatacaggagagaaaccatacaagtgtgaagtatgtgaaaaggctttccgttctctgtcatcactttctaaacacaggagaattcatatgggagataattattataacagtgaattatgtagcaatgccttcgtttatccttcaagactttctaaacataagaaaatttgtagaggagagaagccctacaaatgtgaagtatgtggcaaggccttccatgtttcatcactgctttctaaacacaggacaattcatacaggagaaaaactctataagtgtgaagtatgtgggaaggccttctattatccatcaagactttccacgcataagagaattcatacaggagaaaagccatacaagtgtgaagtatgtggaaaggcgttctgttttccaccatcactatctaaacacaagagaattcacacaggagagaaaccatacaagtgtaaagaatgtgggaaggccttccgatttccatcatcactatctgcacacaagaaaattcatacgggagaaaaaccctacaagtgtgaagtttgtggcaaggccttccattatccatcattactttctaaacacaagataattcatacaggagaaaaaccctataagtgtgacatatgtgaccaggcatttcatgttccatcaaaactttctcatcacaagataattcatacaggagaaaaaccctacaagtgtgaagtatgtggcaaggccttccattattcatcactactttctaaacacaagattattcatacaggaaaaaaaccctacaagtgtgaagtgtgtgacaaggccttccattatccatcaagactttttagccatacaaaaatgcatactggagagaaaccatacaagtgtgaagtgtgtggaaaggccttctgttctccatcatcactttctaaacacaagagaattcataaagttgaaaaagcctacagttgtgagatttgtggaaaaaccttctgcATTCCATTATTACTTTCTAAACACAAGATAATTCATACAGGTGAGAATCACTATAACAGTGAGGTATGTAGCAAGTTCTTTGTTTATCCTTCAAGACTTCCTAAACATAAGAAAATTTATAAAAGAGAGAAACCATACAATTGTGATGTATGTGGGAAGGCCTTCGATTATCCATCAAGACTTTC
This mutation is a 1775 bp deletion beginning at Chromosome 13 position 67,592,123 bp and ending after 67,593,897 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
52506 | CAC AGA ATG CAG AGA ATA TGG A | Common | A | |||
52507 | TTC ACT CCT GTA GGG CTT CAC | Wild type Reverse | A | |||
52508 | ACA AAG AAC TTG CTA CAT ACC TCA C | Mutant Reverse | A | |||
52509 | Fluorophore-1 | AGT TTT TGA GTG GTC CAA AGT GT | Quencher-1 | WT Probe | ||
52510 | Fluorophore-2 | ACA CAA GAT AAT TCA TAC AGG TGA GAA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
52506 | 0.40 uM |
52507 | 0.40 uM |
52508 | 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 |