Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr7:6131056-6131169 114bp TGCATCTTGATGGCTGCT CCATGGTAACTGCCAACACC
Mut= 123 bp
Wt= 114 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TCCTAGCCTGCAGACCTGGCCCTCCCATCCTCACTGGGTTTCAGGTGCCACCTGTGTTGTGCTTCTCATTGCCCTCCTACCCTTGGCCCTCGATAGCATTGGTCGGTAGACCTTTATGCAGGCCTGTAGCCTTAGGCATTGGTCTGCAGATCTAgagaaaggctaccagagacaacacccactcggaaggagatcaagtattagggagatggattatgaacacagaaatacttgaaatgccttgtgtggttggtgtcacacgggataaggtgtactggatatgcattggtacaacccagcagtccccaggttggaggaaccccgaaaggaagagctgccctgggccactcaataagctggaatccattggcagcctgcgcatggatgttcacctctgacttgcctcctccttccctcttgctttgcagtggacatcctcatcatggatgacgatgacgtccccagctggcccccgaccaagctgtccccaccccagtctgcacctcctcctgggcccccgcccaggccgcggccaccagcaccctacatctgcactgagtgtggcaaaagcttcagccactggtcgaagctgacacgacaccagcgcacgcacacaggcgagcggcccaatgcctgtaccgactgcggcaagaccttctcgcagagctcgcatttggtgcagcaccggcgcatccacacaggcgagaagccatatgcctgctcagagtgcggcaagcgctttagctggagctccaacctcatgcagcaccagcgcatccacacgggcgagaagccttatacttgccccgactgcggccgcagcttcacacagagcaagagcctagccaagcaccggcgctcccacagtggtttaaagcccttcgtgtgtccgcgctgcggccgtggcttcagccagcccaagagcctggcacgccacctgcggttgcacccagagctgtcagggccaggtgtggcagccaaggtgctggcagccagcgtgcggcgcgccaaggcaccggaggaggccacagcagcggacggcgaaatcgctatcccagtgggcgatggcgagggcattattgtggtgggcccgccaggggatggtgctgcagcggccgcagctctggctggggtggggacgcgggcaacggggacacggtcgcgcagggcgccggcgcccaagccgtatgtgtgcatggagtgcgggaagggttttgggcacggggctgggctgttggcccaccagcgcgctcagcacggggatgggctcggggtagcggttggtgaggagcctgcacacatctgcgtggagtgtggtgagggctttgtgcagggtgccgcactgcggagacacaagaagatccatgcagttggggcgccttcggtctgcagcagctgcggacagagtttctatcgggcgggtggagaggacgatggagaggaccagtcggctggtgcgcggtgtgccgaatgtcggggtggggaggcccggtagggacggggaggcccggggggtggacagggcccctcgggctcggtgggaactaccagtgtgcaagaccacgagggaggcccagagagaatcagaccgaaaggaacagcggccttgtctccttccttactccgtgctgttctgggtgtgaggggacctgcaactattgggttctcaggactgtccacagcctctctgccgacccgccctaccgcctggtgggctggccctttgtaggtttccattctgggagccagagactgagagaactgcaaggatcaattggcactagaccctcaagggaaagaccacccccactacaggtgttgggctgcggtgtcgtaagaaatggagggaatcctggccttatgcatcaaaagggcttccttcccttctagaattttcttttaatcatactgtctgacccgtcccatccattattttttctgccccccatgggtccttatcaattttctagcccaagtcgttcctcttcctcttggatttgcagtagcagaggcgtgaagtagcctcctgttcccagcacggtcttattgagaggtcctggcagtggaaaaagggatgtgggggcaataaatggcattattcaattgtcccagcactgcctgtttcttgagtttgggatggagaagggagagaggttaaacctagccatcctcactttggtgcacacaagtgacaagcaccagatgatgggatgaccatgttggggggcccagagtgtcccaagccagaaagagatgtgtttgccaaggagccccttgctgacctggtacaaggtcactgttgggaaggtagtctgttcctgctgggaaaaggatggcttatggaagacatagaaatagcacacccctctatctctggtccaagttctggtcactcatcagccatatatcagcattctctgcagagcaggctgcatcttgatggctgctactgttcagtgaaggggcggaggcggaggaggaggagGAGCTTGGAGTAGATTGTTTCTGGACCCAATACTGAAATGGTGTTGGCAGTTACCATGGCATGCTTTGTTCCAGATGCTCCTCAGTGCCAGTCCAGTGCATCCCCCAGAACCCTACAGCTGCAGGATCAGAAGGGACCTCCAGCGTTAGGTGTA
This mutation is a 2331 bp deletion beginning at Chromosome 7 position 6,131,115 bp and ending after 6,133,445 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
42657 | TGC ATC TTG ATG GCT GCT | Wild type Forward | A | |||
42658 | CCA TGG TAA CTG CCA ACA CC | Common | A | |||
42659 | CGA TAG CAT TGG TCG GTA GA | Mutant Forward | A | |||
42660 | Fluorophore-1 | CTG TTC AGT GAA GGG GCG | Quencher-1 | WT Probe | ||
43740 | Fluorophore-2 | TGG TCT GCA GAT CTA GAG CTT GGA G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
42657 | 0.40 uM |
42658 | 0.40 uM |
42659 | 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 |