Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr6:58907072-58907168 97bp AGTGAGCAGCCATTCACTTG GGTCCAGAGTGCGTCTGAAG
Mutant= 94 bp
Wild Type = 97 bp
GGGGGGGGGTGTATAAACAGGTATGGAAAAATACAGTCTCAGCCTCGAGCTGGTTCCAGCCAGTTCTGCAGGGGTGGGGGCGGATGCTGAGGGCTGGGTGTCTAAGTGGCAGGTTTTAGAGACCTCGGAGATAGCAGCATAGCAACAATTTGGTGCAGTGTGTGCGCACAACCATCAGCTGATAGTGAGCAGCCATTCACTTGCCGCGAGCcgcgaagcggccgcgcggacgtcaccacagccgtcagcgtttgccgcatcttcagacgcactctggaccagccgcaatcacagctgcaggacctctctggaccagctctattgcagactgcgcaaccaccagaccactgcggcaaacaagcccagctgagccgagcagtagccatggccgaccccgagaagcagggacccgctgagagccgcgccgaggacgaggtaatggaaggcgcccagggtggcgaagatgccgcgaccggtgacagtgccgctgctcccgcggccgaggagccccaggcccccgcggagaacgcgcccaagcccaaaaaagactttatggagagcttgcccaattccgtgaaatgccgggttctggcgctcaaaaagctgcagaagcgctgcgataagatcgaggccaagtttgacaaggaattccaggctctggagaagaagtacaacgatatctacaagcccctgctcgccaagatccaggagctcaccggagagatggagggctgcgcgtggaccctggagggagaggatgatgaagatgacgaggaagaggacgacgaggaggaggaggaggaagaagaggctgcagctggcgcaactgggggtcccaacttcgccaagaagtgagcacagcaagctgacaggtgataaatcccctgccccttttatttccaatgtcggcagactttaaaaggctcaggatttgcccaaaataaaatgtgaatctattctgacattcctaaaattaaattgaatcaagtagattctggctagcccgtttctaatctgacttttatttgtgtaaaacagatgctaaaagacgttttttagaaaaaaaaaaaagcctaattaaacttttttttttctccctgctcttctgtgagaactggacttgagactgttgaaaggttgtcagttagatgggaattaaaagtgcgtcacacgttgaaatccattcatcacactacaccagaacatccaagctaagacagaactcttctcaatgcttaattcttcagtttctttacattttccagcgcagaggaaaaggaacccaggaacgacgtcatctttaagacttttgcttttgcaaacccagacatcagctttacactccagaggagccttggcatggagcaagcctggactgacagcatttactgattatgtggctaggaaaactgccatttcaagttgtgaaaaaatgttttaaatatttgcatttacagaaagaacatggtttcaaaaatgagggtgtattccatgtataatattgtcaacacgtgttcatctgttatggtcccgtgttacctgttttcttggtagtgtttgtttacaaaatcgtaaaaaattaccccaaatgttttaaagtgttaaattaccttatagcattttagaaatataatttacttgaagagatgtagaatgtagcaattctgtaaagcatgtgtatccagtgttgcctagtttgacctttgtgaagtctttttgtcttgtagcttttagcaagtagctgtgaaaaccatcagaactcctcaatgaagctaatgtttggaaaaagtatatacttgaagaacaaacccaagtgtgtacccccaaccccagctcagaaatagaaagggtttaagtttgcttgtattagctgtgccttcattattttgctatgtaaatgtgacttaataaatggtgcaaaattaaattttattgcttgaggacagacgggcataaaggaagggaaaagacatttaatgtaaaggcctctagcttctttgtgggttttgagttgtctgtgaaccaatgttctgtaaagaaacacaaacttaaagttgtttaccactgtggtgttaacaaaacagtattttcaaaaataaaaaaatccttgttattctgactacctgtactgtcatctataaaagtgggtgggaacaatcccttcctcgcagttattatgaagagaaagtgagtaactacgcttacagcccacacagcagcgtctgacacctaggaataatttccagctattattgttttaataaagacaaaagacccagatagaaaattcaactccctcccctgctacacaacagaaaatggtagcattattaatttctaacttggtgattccttgttatcagaaaagaatatggcTTGTAATTTAGAAATATAGGACATTCAAAAACTATCTTGTTTTAGTTTCCTCCATATAAAGCCAAGTTTTAATTTCCTCTTAGACAAGGTAAGATTTTAATGTGACAATATGAATGCACACACACACATGCACAAACAAACCAAAAAATAATCAGACGCCATTCTTTTCCATTTGCTGCAAGGGTTAATCACACTGCAGCAAAATCT
A 2165 bp deletion beginning at Chromosome 6 position 58,904,976 bp and ending after 58,907,140 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44798 | AGT GAG CAG CCA TTC ACT TG | Common | A | |||
44799 | GGT CCA GAG TGC GTC TGA AG | Wild type Reverse | A | |||
44800 | CTT GGC TTT ATA TGG AGG AAA C | Mutant Reverse | A | |||
44801 | Fluorophore-1 | CGC GAG CTT GTA ATT TAG AAA TAT AGG | Quencher-1 | MUT Probe | ||
44802 | Fluorophore-2 | TCA CCA CAG CCG TCA GC | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44798 | 0.40 uM |
44799 | 0.40 uM |
44800 | 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 |