Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr6:29469774+29469932 159bp GGGTGGTGGTTTGTTTTGT CGTGGAGGGAACTGGTAATG
Mut = 129 bp
Wt = 159 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
TTCTCTTCTCTTCTCTTCCTCCTCCTCCTCTTCTTCCTCTTCCTCCTCCTCCTCCATTTTTTTTTTTGTGGTGGTGGGTGGTGGTTTGTTTTGTTAATAAGGCTCTTGTGTTTCTAGACTTTCATGCAGAcacagaggggctcctatttctctctcctctgcctctcctccacaggggcagcttaaagaacacactgacttgcaccctggtcttcattaccagttccctccacgagctgatgtgtggactccaaccctgagctgtgtggttctgagctcctatgtgtagttgcttcatccaagaaaagagaaggctgaaggccttatacctccggccactgtcaattcctcttcttctcccccaacaggcaatttctaaacagggatgacattggcatcattctcatcaaccagtacatcgcagagatggttcggcacgccctggatgcccaccagaggtccattccagctgtcctggagatcccgtccaaggagcatccctacgatgcagccaaagactccatcctgcgcagggccaagggcatgttcaccgctgaagacctgcgctagggctgctcagccctcagccctcctcatcaccaggcctctcctggtttgccatgctctatttaagtgtcaagccttggtttccagcccctcccttcctcttcactgagtggctcgtaagctgttggggttcggttgttcaggtctaggctgggtagagaaagaaagcccaactgtttctccactgcctcctccctgtgctgttaatagtatcgttgttgatattaaaggaattattctctccaaatgggtactggtatggatacgtgaaggttgcagcattgggggacattagacttgcaaatatggaaaagcaaggtgaagaataattggtggggggaataattcaaggactggtcacctggatcagagactgccttcagcgatggaggacaaagtttgagcccagtctttctacctttgctgcttcacctagggtcttcacggtgctgggaggggcctgcccAAGGGACAGAAGCAATTGCCTAGGATTGTCATTGTCATTCTAGATGAGTGTTCTCAATCTGTGGCTCTCCAAAACCACAGGGGTCCCCAAGACCATCAGAGATCACTGATATTTAAATT
This mutation is a 899 bp deletion beginning at Chromosome 6 position 29,469,831 bp and ending after 29,470,729 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
49998 | GGG TGG TGG TTT GTT TTG T | Common | A | |||
49999 | CGT GGA GGG AAC TGG TAA TG | Wild type Reverse | A | |||
50000 | TTT TGG AGA GCC ACA GAT TG | Mutant Reverse | A | |||
50001 | Fluorophore-1 | TCT GCC TCT CCT CCA CAG GG | Quencher-1 | WT Probe | ||
50769 | Fluorophore-2 | AGG GAC AGA AGC AAT TGC CTA GG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
49998 | 0.40 uM |
49999 | 0.40 uM |
50000 | 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 |