Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr13:92683100+92683210 111bp CAGGGAGTTTATTGTTTCTACTAGGG TTTCCCATAAGGAGGCACAC
Mutant= 100 bp
Wild Type = 111 bp
GTCCAGCTAGAAACTCTAGCGGATAGGTTGTTCTTATTCAGTCTTGGTGCTGATTTGCACATGGCTGTTTTTACCATGCATTTGACTAAGACTGAACACTCACAGGCAGCTCCTCTCagcctagttctaacaggcaacttttgcatcttctctctcagattcccttttttgaagagttctgtaaaaagactcaagccggtggtgacgcctgtgagaacctggtagggtattctgcggtgtacagagtctgctttggaatggcttgtttctttgctctgttttgcctgctgaccttaaaagtcaacaacagcaaaagttgccgggcctacattcacaacgggtaagtctcttgttcttccaagggtgtcctagggtctgctcgtcctcactgactgacgttctgctagttgacatttgacactgatgtggggaggatgggctgtgtgtcactggtagcctaagagcggcagtgcctggtgccataataagaaattcagtatacacagggagtttattgtttctactagggtcttcccctgagtcatttccatcctaccgTGAAGAAGAGTTTTATACAATCAGAGGCCATTCCAAGTGTGCCTCCTTATGGGAAAAGCCACTCTGTGGCACTCCATCACTTCCCCTTGATGTGGGAAGAACACTACCAACAACAGAAACATGAGCTAGCAGGCGGCCTTACAAGGA
A 441 bp deletion beginning at Chromosome 13 position 92,682,714 bp and ending after 92,683,154 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
45979 | CAG GGA GTT TAT TGT TTC TAC TAG GG | Wild type Forward | A | |||
45980 | TTT CCC ATA AGG AGG CAC AC | Common | A | |||
45981 | CCA TGC ATT TGA CTA AGA CTG AAC | Mutant Forward | A | |||
45982 | Fluorophore-1 | TCC CCT GAG TCA TTT CCA TC | Quencher-1 | WT Probe | ||
45983 | Fluorophore-2 | AGG CAG CTC CTC TCT GAA GAA G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
45979 | 0.40 uM |
45980 | 0.40 uM |
45981 | 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 |