Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr7:45354619-45354713 95bp CCCTCCTCCTATTCCAGGTG AAGGGATGAGAGTTGAGAAGGA
Mutant= 98 bp
Wild Type = 95 bp
Wt Sequence: gggcacagtgaggatatctcaggggaggggctaaaatgggggcggggcttagatgaggagccgggttgagtgaGACATGAAGGGTGGGGCTCAACTGAGGATCAGAGTAGTGGGTGGGACTGGTTTGGTTGAGATGAGGGATGGGTTAAGGAGGGACTAAACAGGAATCAAGATGAAGCAAGGCTTAGATGAGGGGCATGATTTCGAACAAGGGGAATGATCTCAGAAAGACCCGCTGGACAGTCTCTGCCCTGTTTCCCTTTGTTCAGGCCCGGCAGAGGGAGAAAATGAACGATGACCACAATAAGCGACTGTCAGAGACGGTGGACAAGCTGCTAAGTGAATCCAACGAACGGTTACAGCTTCACCTCAAAGAACGTATGGGGGCACTAGAGGAGAAGGTGCGCCCCCTCCCACCCCTGCCCCAGACCTTTGTCAACCAGGGGTGGCTCCAAATGCCGTCCTAGGGCCCATACATCTTCAGATCTGTCATTTGAGACCTCCATACCCACAAACTCTGCTCTGTGACTGAAGGCACCATTTTGCTTGGCCATTCCAAGAGCGGAGTTTTCCTAGACCACAACCCCTGCAGCCTGCCTTTGACCTTCCATTTCACCCCTGTCCCCACAGAACTCACTGAGTGAGGAGATTGCTAACATGAAGAAACTTCAAGATGAGCTGCTGCTGAATAAGGTGAGGACCCGGAACTTAATGGGGAGTTATCGAGTGGCCTGTTCCTCATAGCAGTGACATCGAGCCCCATACCTCTGCCTCTCTCCACAGGAGCAGCTCTTGGCTGAGATGGAACGGATGCAGATGGAGATCGACCAGCTCCGGGGAAGGCCGCCCTCCTCCTATTCCAGGTGACAGAAGTGCTTCCTGCCCTTTCTGTCTGTGgcaagctcctcctttccgctcctccttctcaactctcatcccttattagtttatttattttatg
Deleted Sequence: GACATGAAGGGTGGGGCTCAACTGAGGATCAGAGTAGTGGGTGGGACTGGTTTGGTTGAGATGAGGGATGGGTTAAGGAGGGACTAAACAGGAATCAAGATGAAGCAAGGCTTAGATGAGGGGCATGATTTCGAACAAGGGGAATGATCTCAGAAAGACCCGCTGGACAGTCTCTGCCCTGTTTCCCTTTGTTCAGGCCCGGCAGAGGGAGAAAATGAACGATGACCACAATAAGCGACTGTCAGAGACGGTGGACAAGCTGCTAAGTGAATCCAACGAACGGTTACAGCTTCACCTCAAAGAACGTATGGGGGCACTAGAGGAGAAGGTGCGCCCCCTCCCACCCCTGCCCCAGACCTTTGTCAACCAGGGGTGGCTCCAAATGCCGTCCTAGGGCCCATACATCTTCAGATCTGTCATTTGAGACCTCCATACCCACAAACTCTGCTCTGTGACTGAAGGCACCATTTTGCTTGGCCATTCCAAGAGCGGAGTTTTCCTAGACCACAACCCCTGCAGCCTGCCTTTGACCTTCCATTTCACCCCTGTCCCCACAGAACTCACTGAGTGAGGAGATTGCTAACATGAAGAAACTTCAAGATGAGCTGCTGCTGAATAAGGTGAGGACCCGGAACTTAATGGGGAGTTATCGAGTGGCCTGTTCCTCATAGCAGTGACATCGAGCCCCATACCTCTGCCTCTCTCCACAGGAGCAGCTCTTGGCTGAGATGGAACGGATGCAGATGGAGATCGACCAGCTCCGGGGAAGGCCGCCCTCCTCCTATTCCAGGTGACAGAAGTGCTTCCTGCCCTTTCTGTCTGTG
824 bp deletion beginning at Chromosome 7 negative strand position 45,355,486 bp and ending after 45,354,663 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
37874 | CCC TCC TCC TAT TCC AGG TG | Wild type Forward | A | |||
37875 | AAG GGA TGA GAG TTG AGA AGG A | Common | A | |||
37876 | CAG GGG AGG GGC TAA AAT G | Mutant Forward | A | |||
37877 | Fluorophore-1 | TGC TTC CTG CCC TTT CTG | Quencher-1 | WT Probe | ||
37878 | Fluorophore-2 | CTT AGA TGA GGA GCC GGG TT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
37874 | 0.40 uM |
37875 | 0.40 uM |
37876 | 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 |