Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr11:20727690-20727833 144bp TGGTTTGGTTTCTTGTTTGG CATAACAAGGCTTTTCAAGTTTC
Mut = 157 bp
Wt = 144 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
CACACTAACACAGGAACCTGAGTTTTATCCACTGGAACCTTAGGAGTGAATAGGGAATAGGTGGTTCCCTAAAGCCTGCACATTCCAGACCAGTAGGACATCAGGTCTCAAAAATCAAAAAGGTGGACGGAGCTCGAGAGACAGTATGTGAATTGCACTCGGAGGTACATATGTACCTGTATACACACATGGAAATACATATACACAGAGTAGAGCAATGTAATTTTCATTGTTTTGGCGGTGGTGGTTTGGTTTCTTGTTTGGTTTTTGAGACTAATGcccaggttgactggctcaggatgaccttgatgctcctcctggcactccctgagtgaccttgccagataactttgtattgttttaaagaaacttgaaaagccttgttatgtaatctgcctttgaaaataaacaaaactaatgcttttcctttttttaaaggcataaactaactactggaaagctgcggaataatggagccagacatcattcgaatgtactcttcctcccccccacctctagacaacggagcagaggatgatgaggaggatgagtttggggagtttggtgggttttcggaagtcagcccttctggtgtagggtttgttgattttgacacaccagattatactcgtcccaaggaagactttgtaccttcaaaccactttatgccaattcatgaatattcagaagatgtagatagccttacaagctttaagtctgttcagaatggtaatgataaggatatcactgctgaactttctactcctgtgaaaagtcaatctgatgttgtactttctaccaccagcaaagaaatgattccatctaaaactttggatccttccatcgatggtatggaaagcctggaagacttagataaagtagtggtgcagggaccaagcactgggcaactcagaagtttctctcctggagattttagaactgataagaacattgttcaccaaaccaagcagttagagagctgcaacggtgaaaagcctccttgtctggaaattctaacaaacgggtttgcaggtttggaaactgtaaatcctcagggaacagatgacctggacaatgtggcagattcaaagggatcgaagcctcttaacacttgtggcaccgagtgcattttagagtctgcagctagtcatgctacggaatttgcagatttttctacattttcccaaacagaaaggacacaactggaagaaatagaatgtccagttttaaatgatggtgatacactgaccattcagggaaacagcaaaggccccagagtcaaggaactaaactgtgtgaaagaagtgactttggatggaagctttgaggatacggggaacactgaaagagaacatcaggtctgcgtctcagaaatccatgccgtagctgacagagggctcagtgttgaaaaacaagaccttcaaacactgcaacaggatgagtttctaaattcaaggattcagtctgaggcttggagcttggtagactcaagtgaaaattcagaagccattacaaaggagcggtgtaaaatggaaaaaaatgacttatttgcctctaagtgtgctgacttatccatggattctgttaaaacttcagacgttaatgaaattggttcttccaaagaagaaaacagaaagctgactaatcccaaaagcccagaccctgatcccacaggacaaaatgctttggatgattctgctgcaagcatgaaaaatggtgacagtgggaatggctttgtgacttgtcatgataccaatgaggatgattttggtgactttggtacagccaatggcaccactccaccctttgttactagtactcaagattcaatgagtgacgtcacttttgaagactcctcagagcattttctacatcttagtgaaccaggtgatgattttggagaatttgaggatacaaatgctgtttcttgtcaagaggaaatgagatttactgagtcagacctaagacagacttctgatggcttatcagaggaatgtccattggcaggagagtctggtggaaaagacagtaaacctgactcaaaactgaaaaatggtcaagacagtgaatttggagattttgattctgtgccaaatactcaaggtagtgcttttcaagactcggatgattttgcagacttcagttcagcaggtcctagccaagctgtagattggaatgcttttgaggatgaacagaaagatggttgttcttgggctgcctttggggatcagcaggagactgagtctcatcatctaaaggaagtctggcagtcacagaggacagatgaaactatgggcactctgggaacccctaagatgcacagtgtttcttcagcagcttccaaaggagcagttgctagtggccatttgcaggaaccaggcacttcagttcaggtatgtacttactgcctctgatttgtagaacatattaattgccaaggagacttctaatacagctttaaacaaaattacctgagtatctgtcaaagaggtttctgtgtgatatatgaggtttgttggtctggtttgcacattagttagatttttcacaaccctttgaaaaattgtaaaactgctacacagggagtgtcttaactagttaacaggaatttatcaatttaaattatccatggaTAGTATCTGTTTTAAATAGTAGCCATTTTTCCTTCTGTTTTTATCTTTAATAATTATTTCAATTAACAGACTGGTGAAATACTTTCAGTTTAGAATGGGTTTCTGAGCAAAAGACAACTGGGAAAACCCTTTTTTTGTTTGTTTGTTTGTTTTTTGTTTTTTTGAGACAGGGTTTCTCTGTGTAGCCCTGGCCATCCTGGAACTCACTCTGTAGACCAGGCTGGCCTCAAACTCAGAAATCTGCCTGTCTCTGCCTCCCGA
This mutation is a 2354 bp deletion beginning at Chromosome 11 position 20,725,445 bp and ending after 20,727,798 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48889 | TGG TTT GGT TTC TTG TTT GG | Common | A | |||
48890 | CAT AAC AAG GCT TTT CAA GTT TC | Wild type Reverse | A | |||
48891 | CCC AGT TGT CTT TTG CTC AG | Mutant Reverse | A | |||
48892 | Fluorophore-1 | CAG GAT GAC CTT GAT GCT CC | Quencher-1 | WT Probe | ||
48893 | Fluorophore-2 | CAA TTA ACA GAC TGG TGA AAT ACT TTC A | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48889 | 0.40 uM |
48890 | 0.40 uM |
48891 | 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 |