Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr7:127707637-127707737 101bp AGCGCACTTACACACACACA AAGGTGCTGCATCAGGTTG
Mut = 100 bp
Wt = 101 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
CCAGAGGTCTACAAAGCGTGGTCCGGAGTGGTGCTTTCCCTGAGCGGGAGCCCTCATCTATGCCCCGccccctgaagcttttgacactctagcaagcttggccccgggtcttcattgccagagtccctggccaggccttggtggcgccctgctgagtgccccaggtgtctgcaaaaccgcagctccaagttatatccgtttctgctttgccctggggtttgagggctctttctgcttctgtgccagtgttagcgaagctgtccctaccctagcacacctccctgacctccagtctgtggggctcatccttgggcctccctgatccagtctcctgacctcccacagggagaagcgctgggtgactgtgggagacacttcccttcgaatcttcaagtgggtgcctgtggtggatccccaggaggaggtgagcaagccccaacccccaacaggccttgcatctgtgcagcctcaagcaatttccctcccagccttggcctccaccttcagtcggcttggagactccagtgtggagcccaggcagatggtaacgattattagggggcgcgagcgcacttacacacacacacacacacacacacacacacacacacacactttctccaagttcaaggcctaactcaatctacacaacctgatgcagcacctttcgcttctctgagcctaggtttccttattcgtattgaagggctttgtaagagctccctagacaactagctctgatcttacctctctaacccccaggagaggcggcgggcaggaggcggggcagagagatcccgtggccgggagagacgtggtaggggcaccagtcccagagggggaggccccctcatcctactggatctcaatggtatgtagagattctggaatggagttgggtgggggggactgaaggaatcccctggggatctctggctaattaacgcgcggcttttgcttcatcagatgagaacagcaaccagagtttccattctgaaggttcattgcaaaagggtgctgagcccagccctggggggacgccccagcccagccgccctggatcaccaactggacccccagaagtgattactgaagatactcagcccccacaattgggtcaggagagaggtagggccctgtggctctcaggagccctctcatcaaagttgaacccaccatcctgtccttgtttcttatattatgtcctctcacccggtcccgcctccctcagggaatcttccctgaccctggcagagatcacctccagctgtgcctcaatcctccgGCTTCTTAGATCTGTAGGGAGCATATCACCAGCTGACTTTTTTTTTTTTTTTTTGCAGATTTATTTCTTTGCTTGCTTATTGTCTTTTTCTGTTATCA
This mutation is a 1218 bp deletion beginning at Chromosome 7 position 127,707,019 bp and ending after 127,708,236 bp (GRCm38/mm10). There is a 26 bp insertion (GGGCCAAGCTTGCTAGAGTGTCAAAA) at the deletion site.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
49916 | GGA GTG GTG CTT TCC CTG AG | Mutant Forward | A | |||
49918 | TGG TGA TAT GCT CCC TAC AGA | Mutant Reverse | A | |||
51664 | Fluorophore-1 | CGG GGC CAA GCT TGC TA | Quencher-1 | MUT Probe | ||
51946 | AGC GCA CTT ACA CAC ACA CA | Wild type Forward | A | |||
51947 | AAG GTG CTG CAT CAG GTT G | Wild type Reverse | A | |||
51948 | Fluorophore-2 | ACA CTT TCT CCA AGT TCA AGG CC | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
49916 | 0.40 uM |
49918 | 0.40 uM |
51946 | 0.40 uM |
51947 | 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 |