Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr7:127083522+127083666 145bp AGAAGGGGTTGTTGGGTGTG TCTAACAGGAGGTGGGTCAGA
Mut= 157 bp
Wt= 145 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletions in lower case):
GTAGTAGGATAGGATATTGTGGTATTGCTTCTATTATCTTAGGGTGGTGGCTCTGGTGCTCATTGTAATTAAGCATCAAAATGAATGCTCTGTTGTATGCTGTGGTTGAGTCAGAAAAGAAAGTCATATGGCAAGTGATGACAGAGGCATCTTTGTAGAAACTAGATCTAAAGAGGAGAAGGGGTTGTTGGGTGTGGGAGGCATCTCATCCcgagaccggattctctgcagggagtccctgggggccaagttctaccatctcccatgttggagaaccaaggccacatcaccacccaggcttctgacccacctcctgttagagctagcacctcccaccccctcactcatctccccacttctctccccagctcttcacggccgccccccaaggccccggcccctcccgtggctcagcctcccccctcatcatcctcttcgtcctcctcctcctcatctgcctcctcctcgtccgcgcagctcacccaccggcccccgacgccctcactgcccctgcctctgtccacccacagctttcctccccatgggttgcgcccaccaccaccccaccacccttccttggtctcccctggccccaccctgcccccacccccacctctgctgcaggtgccagggcaccctggggcctcagctgctaacgccctttctggtgagtttggggtcctggcagggtggagtggtggggggccatggccccaaggatgggctcagttggctttggcaggaaccaagggcctgtggagggagtggctcaaggctaaagggaaggccagtcaccttggcccaaagaggctgactcggtggcgccagatattaaagccttcttcccctcccctcccctcccacagagcaggacctgatcggccaggacctgaactcacgctacctgaatgcccagggtggccctgaggtggtgggagcagggggctcggcccggcccctggccttccagttccaccagcacaaccaccagcaccagcacacccaccagcacacccaccagcacttcactccctaccccccgggcctgctgcctccccacggccacatggtgagctcctccttgggctgctgctgaggcctaagggaaagtgtctcctacagggaaaggaagaccctggccccctggctgccagcttactcttcccttttcttccctagtttgagaaatacccaggaaagatggaaggccttttccgacataatgtgagtgtgtgtgagggtgtgtgaagtgtgggaatgtatgtatcagtggagtccagtcttcacctccaaaagcacgaaccctgagcttgagaaagctccttgggggtgttagggtggaagccaaagaactaatgggtagagcagacctgggctgcatctccactcccttcttggactagtgcccttctctccacagccgtacatggccttccctcccgctgtgcccgggctccctccgggtctcccaccagctgtctcctttggctccctgcagggggccttccagcccaaggtgagctcctaatccagacaactactacctcccatcccttataaacatcctggaaccctttacctgtttcccattccccaaaatgtttctagcccccattctcccttctggacccaagtttttgcaaagccacgatccctctcttgaccctttttccagcttagttctagacccctttttaacttggtactggctctgctgtctgatccctacactccccttcactcccctttcccagaacacgaaccctgagctgccaccacgactggggccagtgctgagcgggctaccccagaaggggacacaggtgaggggggcagggcaggtcctgggggagttggaaagtatgctgagggaaagcagaattgacgagagagggtgagttcttggttcttcccttaggtgtctaaagagagaacttttgtctaacaggtctaccccggCATGGGAGTAGAAGGTTGATGATATTAATAAGAAGAATGAAGTAATTAATAGAGCAGTAGTTTTGTATTTTAGTTTTCTTTGTTTGGTGTGATTCTAGATATCAAACCTAAGGCCTCACACATGCATCTACTACTGAGTTATAGCTCTAGCAAGACTGATTTTTTGA
This is a 1748 bp deletion beginning at Chromosome 7 position 127,083,557 bp and ending after 127,085,304 bp (GRCm39/mm39).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
58833 | AGA AGG GGT TGT TGG GTG TG | Common | A | |||
58834 | TCT AAC AGG AGG TGG GTC AGA | Wild type Reverse | A | |||
58835 | TGT GTG AGG CCT TAG GTT TG | Mutant Reverse | A | |||
58836 | Fluorophore-1 | CGG ATT CTC TGC AGG GAG TCC | Quencher-1 | WT Probe | ||
58837 | Fluorophore-2 | AGG CAT CTC ATC CCA TGG GAG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
58833 | 0.40 uM |
58834 | 0.40 uM |
58835 | 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 |