Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr4:155840675+155840758 84bp GGGTAACATGGCAACAAACC AACGCTGTAGGTATTTTTCACC
Mut= 81 bp
Wt= 84 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CCTGCAGAGAGTCACATCCTTTCCTACTTCTCGCTGCCAGATGTCTGGTTTCCCACTCACTCTCCCAGAACTTGTTAAGTGGGTAACATGGCAACAAACCTGCCATGACTGCAGGAGGCCCcaagtggtcctggggtgcaaaggtgaaaaatacctacagcgttattcctcctcttcttccaggctctgcagtcctgtcctcaggtcagtatcccctatccagctgtgggccagaccccttcagggtccactcctgtcctagtcctgcctcctctcttgacttctgccactgactcccccttcccgacaagggccttcagggaccgcagcagccagcagctctctggtgtctgagtctgtggtgagcttggcagccggaacccaggctgtgctacgctgccagagcccccgcatggtgtggacccaagaccggctgcatgatcgccagcgcgtggtccactgggacctcagcgggggcccgggcagccaacggcgccgacttgtggatatgtattcggcgggtgaacagcgcgtgtacgagccgcgcgatcgcgaccgcctcctgctgtcgccttctgctttccacgacggcaacttctcgctgctcattcgcggtacactgtagaaccgtagtgggctgaaactttggcagaaactgaaaccttggcggcaggggtgagggtggggtggaggagtggggaggtgtggtgggtagggggagggagctgagaggccttcctaaggttggagccttggatgggcggggccctagaagggattggccgattttaggggaggggccatagagaggacgggttccctcaatctaagggtatcttctgccaagcagctgtggacagaggcgatgaaggggtgtacacctgcaacctgcaccatcactactgccacctcgatgagagcctggctgtgcgcctcgaggttacagaggatcgtgagtgtgtcccttcctctccttcgctatttccatccctatcctccttacccccaatcttccccctcctcacctctgcgaggtgatctgactgcctttctcaacgcatccgcagccctattaagtcgcgcatactgggacggtgagaaggaagtgttggtggtggcccatggcgcgccggcactgatgacctgcatcaaccgtgcgcacgtgtggactgaccgccatttagaggaggcgcagcaggtggtccattgggaccgacagctacctggggtgtcacacgaccgcgccgaccgcctgcttgacctgtatgcatctggcgagcgccgcgcctatgggccacccttcctgcgtgatcgcgtgtcagtgaacaccaacgcttttgcacgcggtgacttctccctacgcatcgatgagctggagcgagctgatgagggcatctattcctgccacctgcaccatcactactgtggcctccacgagcgccgagtcttccacctacaggtcacagagcctgcctttgagccaccagctcgtgcttctcctggcaatgggtctggtcacagcagtgctcctagcccaggtgtgtgtgtagcctgccggcctgagaagaggtcctgttgtgacctagattgctaattgctagaggtattgccacggctgcctgcctgcctgcctgcctgccttccttccttccttccttccttccttccttccttccttccttccttccttccttccttccttccttcctttcctttccttccttccttccttccttccttcttttccttttcctcctcctcttcctccttccttctcctcctttttttttttttggcttttgtttttttgagacagggtttcttctaggctgtcctggaaccaggctgggctcaaactcaccgagatctgtctgcctctgcctcctgagtgatgagattaaaggcatgtaccaccatcacctggcttgcagaggttttctgatgcttgctacttacatctagatcccaccctgacccgaggccacagcatcatcaatgtcattgtcccagaggaccacacacatttcttccagcaactgggctatgtgttggccacgctgctgctcttcatcttgctgctcatcactgtagtcctggctacacgatatcgtcacagcggaggtgaggctgggcccttggaacagaggggcccagattaaccgagaacagtgagagcaacccaataactctcttcacaggatgcaagacgtcggacaaaaaagctgggaagtcaaaggggtgagtttgcctgcccaactctcctcccagacctgtcctgccctggcctcctaaactgctatctccctttaggaaggatgtgaacatggtggagtttgctgtagccacaagggatcaggctccatataggactgaggacatccagctaggtaggggtgagagccacaagggtccaggggaagtacgttttggagtggcacttattattatttttttccccaaccagattacaaaaacaacatcctgaaggagagggctgagctggcccatagtcctctgcctgccaaggatgtggatctggataaaggtgagcacttttacctcctcatcccatatgagactctacccattccagggactccctgccattttcctgagaaggtcttgggaagtcatgccaaatgtctccattgactttcccactgggactaagggacagagagcagacattagccattttcttggcttccagagttcaggaaggagtactgcaaataaatggaccctgagcttctggctgggccagcagctctgtatcaaaggacatctccctgaccctcctgcggtattcctggctcttctcagcggctggtccgacttacctagaaacttggcagagcagctgcctgtactttgcccttcctagaatcgccacccctcatcttggtgagcaactgtgggttccctagagactctggtatagtacgattgctgcccttcacctgtgcccactgatggttgtacccccaacttaaacacaacaaagatcccttgttaatatccaccaaatgcaaagtccctcgtggcctcttactgctagggtcaggaagacacttaaaaattccagttaagactccctagccaccagttaaacacattagccattgtcctggggggtcttcctgagctgcattgtgcctgtgtactgttcagagccctgctgttataggttctgactcatgggcccgccttgctgctttgggcaacttgaggctagcccagggccctttctctgcttctgattcctttctgccgaatgcctcccaagagctacaccagcagttactgggtaccgtatgactcttggccttgacatccctccctaggctggagtctggggttggggccccatttgtcctctgttttggctgaagatggggcgaagatttggctgagtggcctatgctgtcacatcaaacagctatcatttactcctacttgggaagttttcatgtgacaataaaagatacatctgatttttagtcttggacctgctgctctccttctgttcaggaaggccaagtctgggttttgggaacctccagctgctcccataacctgatccccttgggggaaggtctacgtttgtgtccttctagggtcagatgtcctatgacacaggaacctgttttccatggtgaagggctttcagctccttttttgaagtgtgtgtgtgtgtgtgtgtgtgtgtagggagggggtggtatacaatggtgGGAGGGAGGTTGGCCCTAGAAGGAAATGCTGTTAGCCCAATTCTTTGAGAAGCAAATGCTGAGATGAAGTAAAAGGAAGAAGCAAAGATAGGCAGGCATGTTCCCCAAGGATACAAATCTGAAGA
This mutation is a 3613 bp deletion beginning at Chromosome 4 position 155,840,716 bp and ending after 155,844,328 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
42891 | GGG TAA CAT GGC AAC AAA CC | Common | A | |||
42892 | AAC GCT GTA GGT ATT TTT CAC C | Wild type Reverse | A | |||
42893 | TTG GGC TAA CAG CAT TTC CT | Mutant Reverse | A | |||
42894 | Fluorophore-1 | TGG TCC TGG GGT GCA A | Quencher-1 | WT Probe | ||
42895 | Fluorophore-2 | AGG GAG GTT GGC CCT AGA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
42891 | 0.40 uM |
42892 | 0.40 uM |
42893 | 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 |