Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr9:111635117-111635238 122bp CCCATTTCAGGTATCCACCA TGATGGGTCCTGTGACTCCT
Mut= 125 bp
Wt= 122 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GGTGGGGCCTAAGAAAGCAAGGCTGGGAAGGAAGATGGTGGAATGGGTCATGTGGAGCCTCTCCCTGCTCACTAGCTTATGGGATACAGGATGAACTACTGAGTCCCATTTCAGGTATCCACCAGCCTTGGACTGCCCGCCCTGGGGCCTGGCAGAACTTCCCAGgtatacatggggtctagggcagagctggctgcatcccccacaggagtcacaggacccatcataagagaggctgtcctgtcattgccttgcagctccagaaactaaaacggtcactttctttcaagaccaagagcttgcggagcaaaagtgctgacaacttcttcccaagaaccaacagtgatgtgaaaccgcaagcagacctgctggccaaggccagcccaggccccagcccaatagccatccctggaagcccagcgtccatgcccaccaaggctggcctgcaccccggcagcaacagcaagctccacgcctttcaggagcatgtctttaagaagcccaccttctgtgatgtctgcaaccacatgatcgtgggtaagacccctacccccacccctacaggtacaatgtccttgggaaaaggaatgagagctggaggcccaggttcagGGGACCCATAGACCAGCTCTGAGCTTCAGAGAGTTAAATGTAAGAAGGTCAATGTGGGGAGTGGCCATGATGGAGGGGCCTTACCCAAAGCAAAGGCAAACAGGGCTAGGTGGAGGGAGAATTAGTTTGGAATATGGTTGGAACCAAGACAGCAGCTGGCATCGTCCACCAGAAGCCT
This mutation is a 447 bp deletion beginning at Chromosome 9 position 111,634,731 bp and ending after 111,635,177 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46126 | CCC ATT TCA GGT ATC CAC CA | Common | A | |||
46127 | TGA TGG GTC CTG TGA CTC CT | Wild type Reverse | A | |||
46128 | CCA CTC CCC ACA TTG ACC T | Mutant Reverse | A | |||
46129 | Fluorophore-1 | CAT GGG GTC TAG GGC AGA G | Quencher-1 | WT Probe | ||
46130 | Fluorophore-2 | CTT CCC AGG GGA CCC ATA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
46126 | 0.40 uM |
46127 | 0.40 uM |
46128 | 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 |