Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr3:159515386+159515508 123bp TCAGTCCTATACACACATACACACA TTTCCTGAAGGGGGAAAAATG
Mut= 140 bp
Wt= 123 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GACACTTTGTAACTCAACAGTATACTTTGATTTATACAATTACTAAGTATTAAAAACACTGCCTCAATAAAGAAACTGGAGATCCATCAAGAAAGACACCCAATGTCAACTTCAGTCCTATACACACATACACACACATACCAAACAAACAAATCTGTGTTAAAAGTGTTCAACattcctttcagtttgagtacaaactagaacaaaccaaagcatttttcccccttcaggaaaatacagaaaagataaatcatgaaagaataaccaatgaagatctagaaattgcacctgacaaccaagaaataagccaggaagatgttgaagaagtttggagatacgttattatgatatagtaagacaaaaataagcagtatttaagattctgaagtatgtggattattaacttgatttttttatccaaatatgcaaatgaataaaagtattacttttcaaaacttagaatttaaatatgcattatgatttttaataattgcttttggtacagatttagtacagtataaagaaagaagttataccttttaagtcatcactgtagtttgagatttttattggactcctctcaattttgttgagtaagtatatagtaaaagtaaatagtaaatataatccttgaattgttttaagagaaaaataaagtactcttatgtctttgtttgttgcaagtcatgttcttttcctcttattttataaagaaaaccattgtttatactctctttagcctgcaaaccattttaagtttgccatccatagaagaactcttaaacccaaaccaagtaattcctcaatatataatgtacaacatggccaacacaagtaagcatggagtggtcatactacaagacaaatcaggtaggtatttagaaaagaatctggaatcttttcaaacctagtaaccaaaggtgcATTTATAGGTTAGTATAGAAAGTAGAACATTTTCCTAGGACACATTCATTTAGCAATAATGATGGAATTTTTGGAAGTAAATATTATTTATGCTATCACAAAAAAGTATTTAATTGCTTAAATGAATTAATATGACTTTAAAAATATGGCAGTGATAAACA
This mutation is a 750 bp deletion beginning at Chromosome 3 position 159,515,449 bp and ending after 159,516,198 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46187 | TCA GTC CTA TAC ACA CAT ACA CAC A | Common | A | |||
46188 | TTT CCT GAA GGG GGA AAA ATG | Wild type Reverse | A | |||
46189 | CTT CCA AAA ATT CCA TCA TTA TTG C | Mutant Reverse | A | |||
46190 | Fluorophore-1 | CCT TTC AGT TTG AGT ACA AAC TAG AAC A | Quencher-1 | |||
46191 | Fluorophore-2 | AGA AAG TAG AAC ATT TTC CTA GGA CAC A | Quencher-2 |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
46187 | 0.40 uM |
46188 | 0.40 uM |
46189 | 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 |