Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 133 bp
Wild Type = 151 bp
chr19:5364436-5364586 151bp GGGCTGAACACGTCTACTTCA CCACACTCTCTCCCACAATG
Wt Sequence (deletions in lower case): GAGGGGCGATGCGCGGTGTGTCCGGGTGGCGGCGCGTGGAGGGGGCGGGGCGCTCGGAGCGGGGCGGGGCCTCAGGGCGGGGCCTGCGGGACCTGAGAGGCGTGGAAGAGGCCGGCAGCCCGAGTGAGGGCGGCGAGGCTGGCGAGAGCCGCGTGCTGTTCCTACCACGTGTTAGTTCCCAACGGGGTCAGTGTCTGTGTAGCCAGGGTCGCGGGCTGTGAGGGCTTCAGGGCGGACCTTGACTACTGAGTCTGGGAGTCGCTCTGTTGCCTTAGGTGCGCCACCAGCTAGTGCAAGCTGATATATCCAATATACCCAAAACCAGGACCCACGGAAAGTGGAAATTTCCGAATAgccggggatgggttggggggactcaagggagcagccacagtagctgtggtaggatattgcccttgggacttgtggtcttcagccctaacctacttttcttttttttttttttttttttttggcgggggggattcccaggttaagcctgatcacgatgacaacctcccaaaagcaccgagacttcgtggcagagcccatgggggaaaagccagtggggagcctggccgggattggtgacgtcctgagcaagaggctggaggaaaggggctttgacaaggtgtgggggggctgcaaatgccagggaggggtaggagcgaaggttggcaacaggtggtaccacgcagattgggagtgaggcccacacatgtttggggggtacctggggaggcatggtgcctccttcatttttgtgttttgttgagacagggcttctcagtgctaggattaaaggtgtggctcactgtcccctgcccttttgtgtgttttaaatatcacgaaaatgaaactaggcatagtggattgtgcttatgaccctaacacttgggaagttgagctaagaggattcccgtgggatttaatgccagccctgacaacaaaggaacaaaccctgtctttctttttttttttttttcgagacagggtttctctgtgtagccctggctgtcctggaactcactctgtagaccaggctggcctcgaactcagaaatccgcctgtctctgcctcccaagtgctgggattaaagtcgtgcgccaccactgcctggcaacaaaccctgtcttaatcaaaataaaaatggccttgccctctgaagtatttaccacctaaccctccctagcttcagagatctggcctcttcggagtggtatcagcgtggacagtcctgttgctttagcccatgagacacttagccacatgcccttttctattctgcaggcttatgtggtccttggccagtttctggtgctaaagaaagatgaagacctcttccgagaatggctgaaggatacatgtggtgccaatgccaagcagtcccgggactgctttgggtgccttcgagaatggtgtgatgccttcttgtagtgttctctggggatccctcataccccagccacagcacagtttccagagtctgcagcagggcagggctcctccctgtcctccataaaggaaaagatggctgttgtcaccctcacctcctatgtactccagggtctatggggagttctctcccctcagcgtttcaatctttctgggatcgtttcccttgcatgcctttcccttccacctccccctgcagttccgtggcagcagttaccaactttcctgaatggattcctggcccccttctccctcactatctgttggatgccctttggctcctttcctttttttggcaaaaacagtcactgtccttataaaagatttttaaattaataaaatcagtggccttcatacttggtgttctgtgtcagagagcagataggctggggagttgtgtattcccagggctgaacacgtctacttcattcgttatcagtgcagaccgaaactggtgccttctatttcccctcccaagagggagccaccctagccatgtgggctctgggcaggtgccctgGGTGTCCCAATTCCTAAACATTGTGGGAGAGAGTGTGGGTTGGCTTTTTTCTCCTGGGAGCCAGGTAGGCTGAAGGCTTCCAGATGTCAGCAGTCAGTCTAGGCAGCACCTGGTGGAAAGTCAGTGGGAGTCAAAGGTACCAGGAAGCGGGTGGATGGGATGTGGGTAAGAGGTCACCTGCCAGTCATCATAGCATGTGCTCTTAGCGTCTGGCAAGAATGAATGGGTGGGACTTTTGCTGTTTGCCAAGCTACCATAGAAAAGGAAGGAAATGGTTAAACTTGAAAGCCAAATCCTACTTCTATTGTTTTCAAGCTCATATTGTGAGCCAACCCTGAAACTAGTATCTTTGTAAGCACTTATTACATTTTTTATTTTGGAATGGTAAGAGATTTGGAGCTTAGAAAGCTAATTTGCCCTTGAAC
This is a a 1621 bp deletion beginning at Chromosome 19 position 5,414,502 bp and ending after 5,416,122 bp (GRCm39/mm39).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
59518 | GGG CTG AAC ACG TCT ACT TCA | WT Probe | A | |||
59519 | Fluorophore-1 | AGC CAC CCT AGC CAT GTG G | Quencher-1 | WT Probe | ||
59520 | CCA CAC TCT CTC CCA CAA TG | Common | A | |||
59521 | CGC TCT GTT GCC TTA GGT G | Mutant Forward | A | |||
59522 | Fluorophore-2 | ATT TCC GAA TAG GTG TCC CAA TTC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
59518 | 0.40 uM |
59520 | 0.40 uM |
59521 | 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 |