Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr4:99961303+99961413 111bp CTCAGTAACATGGTAGAGATATCCAAG GAAGAAATGCCAGGGGAAGA
Mut= 129 bp
Wt= 111 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
AGTCAGTCCACATTGAGAACCTTCCCCAGATGGACTGTCACACATGCACTGTGTTCTTGCCTTACAGCACCTCAGTGTATGGTTCCAAAGCTACAAAGTTCCATAGGGAAGAAGGGAAAGGAGGGAGGGCGGGCTTTGCTCAACAGCTCTGTTCTGTCTCAGTAACATGGTAGAGATATCCAAGATGaagatgtttctttaagaaagcatctttgctggtccaacagtgtcttcattgtccatgttcttcttcccctggcatttcttcgtgggagctttgtatttatatgttctggatttcctatcctagattggtcgcctggttattgggcagaatggaatactctccacccctgccgtatcttgcatcatccgaaaaatcaaagctattggtgggatcattctgacagccagccataatccaggagggcccaatggagattttggaattaaattcaatatttctaatgggggtgagtattctgtcatcttaaggacaagtagatacagttggataagctagtgcccgaagccctgggctctgattcccatcttaagaaggggttttgcttccctttggagatggtaaagtacttcttagtctacagctgccttctttgtaatgaattgtagctatcatggtattctactttagaaaagtctaactggtattattagctcctagcactttgaaggttgacacaggagAATTGTGAATTCAAGGCCAATCCAAACTGTATAGTGAGAATTTGACTCAGAAACATCTGGAAAAAAAAAAAAAAAAAAAAGAGTCCTGGTGCTTTGTGACTCAGTACTGGGGTCGAGGGATTGCTGAGTGCTGGGTTGTGTTTTCTCATGAACCGCA
This mutation is a 521 bp deletion beginning at Chromosome 4 position 99,961,333 bp and ending after 99,961,853 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
40551 | CTC AGT AAC ATG GTA GAG ATA TCC AAG | Common | A | |||
40552 | GAA GAA ATG CCA GGG GAA GA | Wild type Reverse | A | |||
40553 | TCA CAA AGC ACC AGG ACT CT | Mutant Reverse | A | |||
40554 | Fluorophore-1 | AAC AGT GTC TTC ATT GTC CAT GTT | Quencher-1 | WT Probe | ||
40555 | Fluorophore-2 | ATG AAT TGT GAA TTC AAG GCC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
40551 | 0.40 uM |
40552 | 0.40 uM |
40553 | 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 |