Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 106 bp
Wild Type = 122 bp
>chr8:69864218+69864339 122bp TTTAAGTGATGCCCAGCCAT TCCTCTTCCTGAAACTGGAT
Wt Sequence (deletion in lower case):
TGTCCCAGTGGGTCACTAGGTGTCTCTGATCCAATGTGGAAATAAAGAAACTGGAGGATGGCTTAGAGGTCAAGAGTGTTTCCTGGTCTTGCAAAGGACTGAGTTCAGTCCCCAGCACCTACACTGGGCAGCATACAGTTGCCTGTGAACCCAGCTCTAGGGGTCAGCCATGGACTTCCTGGACTCCCAGGGCACCTGCAGTCACACATGCACATGCCCACACACAGATGCGCACATAATTGATATTTTTAAGTGATGCCCAGCCATGGTGCCAGTGCCACTTGgtcctggctacttttctcttcagagagcacatcctggtctgcctctgttccctctagccgtgagcatccagtttcaggaagaggaccctcccgatgcccagctgctgaggttggataacatgctgctggctgagggtgtgtccaggccagagaagagaggccgaggagcagcagcaggcagcacagcaacaccaggtggctgtccaaatgacaatagcattgagcactcagactacagggccaagctgtcccagatccgacagatttaccactctgagctagagaagtatgaacaggtgatctttctacatgcaagagtttgtcttgtgaatgtgcactggccacagacagatgtggacatgagctccctgctctatggcagagcagggacagcccgccacagtcagtcactgcagaggggctggtgtcactgaaagcgctttcccctcactcctcagagctgacctgagtgtgagctgtgggggtggcagatgtggtcagctgtggagcccactgccccgcaccactggctgctgagtcacaggctggcccatcttttcccaccccaggcctgtcgtgagttcaccacacatgtcaccaaccttctccgggaacagagtagggtgaggcctgtgtcttgcagggagatggaacacatggtgaacaccatccagagcaagttcagtgccatccagaggcagctgaagcagagcacgtgtgaggctgtgatgactctacgctcacggttcctggatgccaggtcaggggctgcgtgccactggggtcccCGAGGGTTGGCCCTTAGATAATAGAAGCAGGATCAGAGTAGGATAGTTCCGATGTTTATGTCATAGTGTTGCAAGCGCTAATCCCATGTTCTCCCTACCACTGAGCCCAGCCAAGCTCTGTCTTGTTTTTAAGTCTGTCCAGAGGGTCTAGCCAAGGCCTCACATAGCTTCTTTATGAGAAGACCCAAAGTACAAACTGCTGGTTTGT
This mutation is a a 788 bp deletion of region Chr8:69,864,255-69,865,042 (GRCm38/mm10)
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
71825 | TTT AAG TGA TGC CCA GCC AT | Common | A | |||
71826 | TCC TCT TCC TGA AAC TGG AT | Wild type Reverse | A | |||
71827 | ACA CTA TGA CAT AAA CAT CGG A | Mutant Reverse | A | |||
71828 | Fluorophore-1 | TCT TCA GAG AGC ACA TCC TG | Quencher-1 | WT Probe | ||
71829 | Fluorophore-2 | TGC CAC TTG CGA GGG TTG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
71825 | 0.40 uM |
71826 | 0.40 uM |
71827 | 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 |