Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr9:63525247-63525384 138bp CGTATGTAGAATCAGTCCTAGCAG CCTGAGGAAAGAAAGTTTTCCAGA
Mut= 129 bp
Wt= 138 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
ATATATTTATTATATATAGAGAGAAGTACTTTTAGAGCAGTTTAATAGCTTCTCATGAGATATCCCCCAGTAATGAGTTTTAGTGACATTTCTTTGACATCCTTATGTACGTATGTAGAATCAGTCCTAGCAGCCATGCcttcccatcactgggtggtaattgatgaagtgttagtaggtatgtactgatcacactcttacagagatcatggaatattttaattctggaaaactttctttcctcagggcatgaagattaggacaccgtctaagactaggaaatcaccatgggattatatattttctgtttacaacggctgtgtagacaggacagccccagacttcttagcattcaaatcacgcttcaaattaatctgggggagtattttttctttcctggaacaaattgagaaatttctcaaggactatgcaatatctgaagccaaaataaaagggaagagtctggtgtccctgcttccagagtttgagctgaagaacaaattaaccagaaatgatgttctggctgtgttagagaacccagttcacattcagatgctgttaaaccttccgggacagaggtacaagggccaagatggaaaggcagaagctgccaccaagatccaagccacgtggaaaagttacaaggcaagatcttctttcatttcttatcgccagaagaaatgggcctcaggtgtgatcgccctggcgtggcttttacattgccacaggacaagactaaagaggatagtgaaggaatcccgacagagacacctggagaacttccgcattcgagcccaggtatgccaggctgccagctgttaaggcaagcctctttttctaaatatttcctgcttgaacaagtgcagtaacttgtctctttatctttccttccctctccagcctccctccccctcagcattctgcatctataattctcctcaggatatgatacacaaactcagagcctggcctgcatgcctgcaaaatctgttataatttttctataaaagaaaatggtccttgttttgactatggtagttgtgttgggaagtcactgttctaactgcattaccttttctatacacacacacaacacacaagcgagcacgagcaaacacacacacacacacacacacacacacacacacacacaccttctgacactttaatttggtaggaaaaaacatcctcacttgtctatgttgctgtcaccactttgtcagaaagtatttaagtttgcagacagcCTTTTAATTAGTGCTCACAATTTCATTTATGTTTGCTTTCTCTTTTTTTCATTCATATTCTGTTAATTAAGTGAGCTGCCTCTAATCTTCCCCTGCCAGTAGCATCATGTAGCCACCTAATTAAATTAATTGGGGAAGATGTTTTAAGTAT
This mutation is a 1108 bp deletion beginning at Chromosome 9 position 63,524,247 bp and ending after 63,525,354 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44689 | CGT ATG TAG AAT CAG TCC TAG CAG | Common | A | |||
44690 | CCT GAG GAA AGA AAG TTT TCC AGA | Wild type Reverse | A | |||
44691 | CTG GCA GGG GAA GAT TAG AG | Mutant Reverse | A | |||
44692 | Fluorophore-1 | CCA TCA CTG GGT GGT AAT TGA | Quencher-1 | WT Probe | ||
44693 | Fluorophore-2 | AGT GCT CAC AAT TTC ATT TAT GTT TG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44689 | 0.40 uM |
44690 | 0.40 uM |
44691 | 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 |