Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr4:152040726+152040816 91bp CAGCCTGTCCTGTGTTCTACA ACCATGCCCTGCTCAAAAG
Mut= 95 bp
Wt= 91 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GCCACTGCCCTTACCCAATGAGCAGCCGTTGCGGGCTCCATGCTGTGTAGTTTGGGTTTCCTTGTCAGGTGTGGCTGGTGACATGCACTCTGAATGCAGGGATGGTTGGATTTTAATGATAGGAAGAACAGCCTGTCCTGTGTTCTACATAGTTCTGGGACAGCCAGagtcttaaggccctcaatcaagaaagaaaaatccttttgagcagggcatggtggcgcacgcctttaatcccagcacttgggaggcagaggcaggtgaatttctgagttcaaggccagcctggtctacagagtgagttccagggcagcaagggctatatagagaaaccctgtctcgaaaaaccaaaaaaaaaaaagaaaaagaaaaagaaaaaaaaggaaaagtccttttgataggtaaatcactagttagggtgtttaatggtttttttcttttcacactagggtttcactttcagtgggatctgccgtgtgacttgcgtctacggccagttggaagtatacggccatatcatcaaccaagggcagcctccccaagatgtcttttctgtctacacccactcttacctgaccatcaatggggttccgtacgcagaacccgagaaaagtgagaaggcgatcaggagggagatccgggccctgctcaagccttacacgaaactaggtaattggctcccgcttccgatgtcaaggctgatgacgaaggcttattatgggctggacgttgcgctgattcctcatggggggtgtcttacgttatgagagttgaagctggtaggtggcacacttcgatggcctgtctccacccaggtgtaccggtgaccagtgacaacctagAGCTTAGCAGTTGGGACAAGACCCTGCAGGCTCTAGAATTGTCACTGTTGCTGAGGTGTCTTGCTCTTCTTTGGCAGATGACAGAAACTGGGTGGTGCGGTACTTCCCTCCTCTGGGCTCCATTATGATTCTGGAACGGATGCAGTCTCGTTTTGTGGACTTCCTGAAGACCTATAAGTGCTCCTCATACGTGCTCCTGCAGGAGGTAAAG
This mutation is a 675 bp deletion beginning at Chromosome 4 position 152,040,765 bp and ending after 152,041,439 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
39032 | CAG CCT GTC CTG TGT TCT ACA | Common | A | |||
39033 | ACC ATG CCC TGC TCA AAA G | Wild type Reverse | A | |||
39034 | CCT CAG CAA CAG TGA CAA TTC | Mutant Reverse | A | |||
39035 | Fluorophore-1 | AGG CCC TCA ATC AAG AAA GAA | Quencher-1 | WT Probe | ||
39036 | Fluorophore-2 | ACA GCC AGA GCT TAG CAG TTG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
39032 | 0.40 uM |
39033 | 0.40 uM |
39034 | 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 |