Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr7:30727524-30727667 144bp AATCCTCGGACTTAGGTCTCAC TCTAGTGTGTTCATTCCTTAGCTTG
Mut= 158 bp
Wt= 144 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GAGACAACGCTTAGGGACTATGCCCGAATACCTTAGAAGGGATGCAAGGCCAGAAAGGCCCTGATGCAACTTTTTCTTACAACTGTTGTAAAATGAGAAAACCTCTTAGTGCGATATTTCACCCACAGGGTGTCGCAGGGTGCTGCCTAGGCATTTTTAGACATCAGATTCCCCcgtgaacttctagacttttagaaaccaggccacttgccctctatctcccaagatcacgtgatcttgccacttacacctagaatgacccctagaacccagcgtcatatcctctacttgcacacttactttccacagtatccacatagttttttgtttgtttgtttcggtttttaatctccctcaccttccagtgacctttctactttctgatctcagatgttgttcttggccactttcttccccacctgggaaggtggcatctatgacttcattggggtaagttgaacaagggaagggcaaggaagagtgcaggagtcggttcacctggtccccgtgtttactcgagcctctacctgacccacaggaattcatgaaggccagtgtggatgtggcagacctgataggcctaaaccttgtcatgtctcggaatgcaggcaagggggagtacaagattatggttgctgccctgggctgggccactgctgaactcattatgtcccggtgtgtggagcaaactggagcccagacttccgggaatggacagctgggtcccacaggggtgctgaggggaggtgatcagaggctgggtgctgggggtgggggagtctgggtactagagactgcctccttttgccttcctcagatgcatccccctctgggtgggagcccgaggcattgaatttgactggaaatatatccagatgagcattgactccaacatcagtctggtatgcagtctagccctagcctacctttatgttgttagccatacttctcccaaaaacttggctccaacttcccagtcccttcctgcctccctgcaggtccactacatcgttgcatcagctcaggtctggatgataacacgctatgacctgtaccacaccttccggcctgctgttctcctcctgatgttccttagtgtctacaaggcctttgtcatggagtgagctgggtggggtatgaggctgggttcaaatgggggtgtgttttctctctccttttcctcattgtgatgtttccccacaggaccttcgtccacctcttctccttgggtagttggacagcactgctcgcccgggcagtagtaacaggactgctggccctcagcaccttggccctgtatgttgctgttgtcaatgtacactcctagcttgacatctcaggtgttggcgaaccttttcatttcctctctccagatttcagtgaagtaaacagtgtttggaaaggtttttgttatgtccatctctctctctcagaaaccgaatcctcggacttaggtctcacagagtctgtctggcttcagaaccagataaataagctggaatgttggccagtcttcctggtttccccatgagaacctgggggcTTGGTAATCATTGAACAAGCTAAGGAATGAACACACTAGAGTTCAGGAGGGGGCTTTGGGGTAGAGGGGCATTCTTCCTCAACTTACAGCTTGGTTTGAGCCCTCTTCCATGGTGAGTCTTCCCAAAACTGGTTACCTCCAACTTTGCAACCCCCCATTTCTCCACTCTAGTCCATCCACCATGTAACAGCTAGGG
This mutation is a 1369 bp deletion beginning at Chromosome 7 position 30,727,564 bp and ending after 30,728,932 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
47837 | AAT CCT CGG ACT TAG GTC TCA C | Wild type Forward | A | |||
47838 | TCT AGT GTG TTC ATT CCT TAG CTT G | Common | A | |||
47839 | GGC CCT GAT GCA ACT TTT TC | Mutant Forward | A | |||
47840 | Fluorophore-1 | AGC TGG AAT GTT GGC CAG T | Quencher-1 | WT Probe | ||
47841 | Fluorophore-2 | CCC ACA GGG TGT CGC A | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
47837 | 0.40 uM |
47838 | 0.40 uM |
47839 | 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 |