Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr16:17777180+17777279 100bp TTGTGGGTTATGTGGGCTGT TACCCCAGAAAAGCTGTACCA
Mut= 101 bp
Wt= 100 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CAGGTTTTAAACTAAGCCTACAGAAAGTTTCAGTTTTCTGTCAGAAGAGGTTGGGGTCGGTTGAGGAAAGATTCCACTTTGCTTCTCCCAGTCCCAGAAGGCCAGCTACAGGGACAGCCAGCTCTGTATGCCTTACTTCTGCTGTCATCCTGTCTTTAATATTCAGATGCAGTCCTCCAGCTAAGACttgagtgggtatccttctttttacagataccagaaattatccacttctgctgtgatttcctcatgtcatgggtagatgaggagaatatccttgatgtctaccggctagcagacctctttgacttgaaccatctgacccaacagctagatacctacatcctcaaaaactttgtggccttctccagaactgacaagtaccgccagcttcccttggagaaggtctactctctcctcagcagcaatcgcctggaggtttcctgtgagactgaagtgtatgaaggggccctgctctaccattactccctagagcaggtgcaggctgaccagatctcactgaatgagccccccaagctcctagagactgtgcgctttcccctgatggaagctgaggtccttcagcggctgcatgacaagcttggtcctagcccactgagggacacagtggccagtgcccttatgtaccaccggaatgagatcctgcagcccagccttcagggcccacagacagagcttcgctcggacttccagtgtgttgtgggctttgggggtattcactccacaccgtctacaatcctcagtgaccaggccaagtacctgaatcctctactgggagagtggaaacacttcactgcctctctggcgccccgaatgtccaatcagggcattgcagtgctcaacaacttcgtgtacttaattggaggggacaataatgtccaaggatttcgagctgagtcccgatgctggaggtacgaaagggcacttaccatcattgcctttattgttagacaactccatggtgcaggaattgtgggttatgtgggctgttcagggtacacgttatcaagggcacatctctacagacCTGCATCTGCCTTTTATATGAATGGTACAGCTTTTCTGGGGTATACCTGTGTCATGTAGTGAAGTCCAGATGAATCCAGACAAAGTGGACCACAGTGAAATAAGGAAAAGGGCATCAGAATTGCTTTCTTGCCTTTTGACCCTGCTATCCAGAAA
This mutation is an 861 bp deletion beginning at Chromosome 16 position 17,776,376 bp and ending after 17,777,236 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44136 | TTG TGG GTT ATG TGG GCT GT | Wild type Forward | A | |||
44137 | TAC CCC AGA AAA GCT GTA CCA | Common | A | |||
44138 | GCC TTA CTT CTG CTG TCA TCC | Mutant Forward | A | |||
44139 | Fluorophore-1 | CGT TAT CAA GGG CAC ATC TCT AC | Quencher-1 | WT Probe | ||
44140 | Fluorophore-2 | AGT CCT CCA GCT AAG ACC TGC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44136 | 0.40 uM |
44137 | 0.40 uM |
44138 | 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 |