Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr3:51493701+51493808 108bp CTAAAGAGCCACAAGCCATTG CACACCTATGACTCAGCACCA
Mut = 85 bp
Wt = 108 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
GGCCCAGAGGTCAGAGGTACTTGATTTCTGCATCCCACAGGTCAGTGGACAACCAACTCCTGAGCTGATCTCTGACCTGCATGCATTCAGCATGGCGTGTGTGCACCTGTGTGCACACGGGATAAAAAAACTAAAACTGCTTTTAATCCCAGTACATTTGACTCAGCATGGGCCTCTCATAGCACTAGGCCATAGGTATTCTAAATGGTTTTTGTGTGTTGTATCCTTTTCCCGCAGTGATTTTTAAAAACATTTTAAAAGCCCATACCTGTTCAGATTTATAATCTGTATTTAGTAGTCACATTGTGACTAGACTAGGATTATGAAGCGTGGACCCTGCAGGTAAAGACGGGCCAGAGAGCGGAGAGGTGTTACGGGCAGGAACAGTGGCTGAGTTGCTCTGTTTGTGCCATTGGACTGAGCTTGTCGACTTTGCTTTCTGTTTTCCTCTCCagatccagttgtgggacacggcagggcaggagcggttcaggaagagcatggtccagcactactacaggaatgtgcatgctgtcgtctttgtgtatgacatgaccaacatggccagcttccacagcctgccggcttggatagaggaatgcaaacagcacttgctggcgaacgacatacctcgaattctggtgggaaataaatgtgacttgagaagcgccattcaggtgcccacagacttggcacagaagtttgctgacacacacagtatgcctttgtttgagacctctgctaaaaaccccaatgataatgaccacgtagaagctatattcatgaccttagctcataagctaaagagccacaagccattgatgcttagccagctgcctgataacagaattagcctgaaacctgagaccaagcctgcggtgACTTGCTGGTGCTGAGTCATAGGTGTGGCCATTGCGGGTCTGACTTACCTGAAGAGATGATTGGGAAGTGTGACAGTGATGAAGTTCGTAAGAGTAAATCCTAACTGTAGTAGATCATTCTGCTGTTATCGTTGTTATGATGCCTTTTGTATTTTGCATCCTCTCAATCAAGTTTGTCACTGAGACAGCACATGGAAAAGGCCGCTTTACAGTGGGGATTGAAAATGAAGACTACCGGGGCCATCTTTCCAGCTCCAGCCAGAAGCAGCAGCTTCAAGTGAGCATGATGGACCTTAAATGGTTGGTCCTCTTCCTCTCTGGGTCCCTGGAGCAGCAGAAGCCATGCTGGGTAGTGC
This mutation is a 428 bp deletion beginning at Chromosome 3 position 51,493,354 bp and ending after 51,493,781 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48622 | CAC ACC TAT GAC TCA GCA CCA | Common | A | |||
48623 | CTA AAG AGC CAC AAG CCA TTG | Wild type Forward | A | |||
48624 | TTG CTC TGT TTG TGC CAT TG | Mutant Forward | A | |||
48625 | Fluorophore-1 | CAG AAT TAG CCT GAA ACC TGA GAC | Quencher-1 | WT Probe | ||
48626 | Fluorophore-2 | TGC TTT CTG TTT TCC TCT CCA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48622 | 0.40 uM |
48623 | 0.40 uM |
48624 | 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 |