Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr4:43496017+43496117 101bp CCTTCCAGAATGTGAATGGAG AATGGAGTTTGAGCCTTGATG
Mut= 100 bp
Wt= 101 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TATTCCAACTACTATGAGGGAGGATTGTAAGTGCAAGGCCCATCTGGACAATTTAGCAAGACTCTACCTCAAAGGCAGCGAGGTATAGGGATGCTAAATAAGAAGGCTGGGGTAAGAGCAATCTTCCCgtccctactggagagtttcacattcccaggagtgctttagcagtctggccatgtgtgggcatcctgacgtttcacttcatgttctgggtaagcagctggactagagacaactgggattctctgaggtggatgcccagagctttctctatggctcctctcttcactggctctgaggtgctggggcctttgctgcagccttgtctttatctgcttccacaacagggcccagatgaggctgctgttctcagagaggaaaaaaacaagaagaaatcctcgcagtcaggtgggttgagcatttagtggtggtttctggcgatggagtagatcaggggactgcaggagtaaccatgccctttccctcactcccagagcaacagctggtgcagctgacacaacagctggcccagacggagcagcacctgaaccacctgatgacacagctggatcccctttttgaacagtgagaatttgtggggtgggtgaggtgggagccaaggagacagactgggcagccccctgagtgggtatttctacagggtgacgactttggttggaacccagagggaacttctagacacaaaactaaagaccatccaccaccttctacaagactgccagcctggcacaggagtggaagttccagaaccaggtaaaaggttgggaggtttctatggctggagagccatggctgtgaaactagagcaagttggtctctccttcacagaggccagcataccctttactgaggacctggggaaagaagaccaagaagctggaaacagtcaggcctgggaagagcccataacctggagcccagaaacacggaacctagctccttcctgggaggtggagcaggggctgaggagaaggtggcacaagacggtgacaaagggtccagcagtaaatggggaacaaccactgaaggtttagtaaaaagagtcccctcgtactgggtgttcctctgtgctttttccactccctgctacacacacacacacggtgcaaaagccaagcactcttcccttattaggaagagagagactgccttccagaatgtgaatggagaagacagagagagagccacagttctgcccttgtGTTTCTGAGGCTACCGAGATGCCAGCCATCAAGGCTCAAACTCCATTTTCTCGTGATTTAGACAGGAAGGCTTTTGCAAAAACAACCCTTTATTAAGATCCAGTGGGGAAATAAATTATAGAACAT
This mutation is a 1098 bp deletion beginning at Chromosome 4 position 43,494,973 bp and ending after 43,496,070 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
45257 | CCT TCC AGA ATG TGA ATG GAG | Wild type Forward | A | |||
45258 | AAT GGA GTT TGA GCC TTG ATG | Common | A | |||
45259 | CAG CGA GGT ATA GGG ATG CT | Mutant Forward | A | |||
45260 | Fluorophore-1 | AGA GAG AGA GCC ACA GTT CTG C | Quencher-1 | WT Probe | ||
45261 | Fluorophore-2 | AGA GCA ATC TTC CCG TTT CTG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
45257 | 0.40 uM |
45258 | 0.40 uM |
45259 | 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 |