Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mut= 80 bp
Wt= 86 bp
Wt Sequence (deleted region in lower case)
GATGTTTCTGGCAGGAGGTTGCCTGGCTCTGTGAGGGTGAAATAACTCCCACTCCATTTGCCTTGCATGGAACTGTAGGTCTTCTCCGAGCTGGAGTCCTACGTCCTGGCACCAGCTCCTTCGCGTACAAGCCATTACTTTCTAAGATTGTGTTGCCCATCCTCCTAGATAGGGGGAATCAAACTCATAAAATGTCATGCACTTCCTTGGTTTGGGAGAGCCCTTAGCCTCCTAACACATGCACACATGCACACATGCACACACACACACACACACACACACACACATACACACTCCTGAAGAAATCCGACTCCTACTCCATCCCTGCTTACTGCCCCCTCGACCACCACCACCACAGACCACCCAGCCACATCTTTCTCTTGGGGACACCAGGCCTctgcatgatggaagtcaggccagcaactgcttctgggtacctggaaagtatggtttccatctgacattatgaatcgcgagtaaggggatcaaagtcaggaacttcttttggaatagagagctgggatgcctacagcaggcagggtgatgtaagtttaaatccaaataacatatgttacctctctacccctatgttgcttcttccctgccacagggaaaaacgccatctttcgggcggtggtctgtggggaacccagacctgaagtgcactggcagagcagcaaaggagacctcagcaactccgccaagtaccagatatcctctgctcccggtagagaggagcacgtgcttcaggtgagccctgcagctgtttctacccatggttcacggctctcttcccagtccctaaaattcttaatggctctggtcccctggtgaagaagagatattcccacagttgggactATTCATCCTCAACCCGTCATTCAAGGTCTCCTGAGCCAGGAGAAGCCCAGATGGATTTGTTGTCATCTCCCTAATAATCAGGAGACCACCTGGACCATACTGGACCACAGTCCCAGGCCTACAGCTTTGGCTTTGGAAAGCTGGTTACTTGCCTCTTACTGTCTCAATTTCTTCACCTATGGGATAGCATAATTATAGTATACACGTTAAAGAATTATAAAGATTAAAAGTTCATGGAAAGGGTCTAGTTAGTGGTTCTAAACCTGTGAGTGTGACCCCTTTGAGGGTCAACCAACCCTCTCACAGAGGTCACATATCAGATATTCTGTACATCATATCATTACATT
This mutation is a 464 bp deletion beginning at Chromosome 1 negative strand position 135,980,217 bp and ending after 135,979,754 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
37258 | AGA CCA CCC AGC CAC ATC T | Common | A | |||
37259 | TCC AGG TAC CCA GAA GCA GT | Wild type Reverse | A | |||
37260 | CTG GCT CAG GAG ACC TTG A | Mutant Reverse | A | |||
37261 | Fluorophore-1 | TCT GCA TGA TGG AAG TCA GG | Quencher-1 | WT Probe | ||
37262 | Fluorophore-2 | TAT TCA TCC TCA ACC CGT CAT T | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
37258 | 0.40 uM |
37259 | 0.40 uM |
37260 | 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 |