Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mut = 93 bp
Wt = 108 bp
Fam = Mut
Hex = Wt
>chr19:42041693+42041800 108bp TCGAGTCACCAGAGTCACCA CAGGGACACATGGTTTCAAG
WT Sequence (deletions in lower case):
GGTGGTGCTGGGGACCGCTCTCTTGCTCGGAGGTGGAGGCCTCATGCTGTGGGCTCACAGGAAGAAAAGGAAGGCAGccactgagactacagaagacaagcctctggaatcccaccgagcaaatgacagctggatcaaatctcacttcagccgcctatccgaggagaggttgcccacctaccgctatgtcagcaacaatggccactcacctgaaagcaggcatggggaagtcaataccaccctccacgtggataccctcaccaccaaacacggagaaaggggcgcggctctgcaccgagattcgtttgccagcaaacagaagatatctggaagctcaatgaccaaagagatgcagagggagtcagggaagtccccgtccatggaggatgacacctgggctgctgtggcagcttgtacgaaggagattgatgccaaggggcatcgcgtggctaactccatgctgcagcgctccacaactcaccgtcgtaaaggccatgcagaatccaggaacatcagcccagaagagctgaaggctctggaggaggtggagataaaactgaaagggaatttcctaactcaccatgaaactggagtagctggggccaaccaatcgcacacagtctatagccagagtcgccatagtaaccaaagccaccattcctatccaagccaccagagcaaccagagtcatcccgtttactccagctaccagggtcatcatccaagtcacttgagcccccagagttatccaagttactcaagtcaccagagtcatccgggccactccaaccaccagggtcattcaggcctctcaagccaccagactcatctgggccactccaaccaccagggtcatccaggtcactcgagtcaccagagtcaccagggtcagccaggtcacccaagccaccagagccataatctgcccaaccgcagaaacCCAATCTATGGCTCTTGAAACCATGTGTCCCTGCCCCCCACAGGGCTGGCT
This mutation is an 863 bp deletion beginning at Chromosome 19 position 42,040,905 bp and ending after 42,041,767 bp (GRCm39/mm39).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
58132 | CAG GGA CAC ATG GTT TCA AG | Common | A | |||
58133 | TCG AGT CAC CAG AGT CAC CA | Wild type Forward | A | |||
58134 | CTC TCT TGC TCG GAG GTG GA | Mutant Forward | A | |||
58135 | Fluorophore-1 | CAA GCC ACC AGA GCC ATA ATC T | Quencher-1 | WT Probe | ||
58136 | Fluorophore-2 | CAG GAA GAA AAG GAA GGC AGC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
58132 | 0.40 uM |
58133 | 0.40 uM |
58134 | 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 |