Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr4:43424380+43424477 98bp GTGTCTGCTCCTTGGGATG CATGGCCTCTGAAGTGGAG
Mut= 99 bp
Wt= 98 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
ACTCTAATTGAAGGCTGTAGTTGGAGCGACTCTGGGCAAGCATGTGGGTGACAGAAGACAAGAATTGGCAGGAGCATGCTGGTCAGTAGGTTAGTGACCAGTAGTCCAGGAGCTActacagggtaaataaccaagtgggccaaggggcaggagcttggccaagaaggcggagttgcttgtagagccaaacaaaacgcaaaggtcagagagggaaagctgtccatgaagagagcagccaacccccggaaaaggaaagacccacgagaaccgtcggtggggaggggcaacttcctcttccccactcaggtgtccgcctccaccaggtgggctagcatctccccaaggggacctgtgacagccacttctccctcacagggctggtaaaagctgtcaacaccgctgtggaccttattgtggcccattttggcacgagccgggatcctggcgtgaaggtatgctgacactatcaagagctgagctatgccctgcagactcggccgtcacctccctctgctctcggggacggaattcggggacagaaatggccgggggacatccaagagggtctgactaagccagactaaggtgaggcttctcacggttgccctatgtatccgcaggcaaagctgggcaacagttctgtgagccccaatgtcggccacctggttctgaagtacttgtgccctgcagtccaggccgtgctcgaggacggactcaaggccttcgtactggatgtgatcatcggccaacgtaaaaacatgccttggagtgtggtcgaggcttccacacagctaggtaggtactagattgcagccctggactccggactgaatttggggttccagaagttagaaatgtgatctccaaacctgccttctccccaggcccatccaccaaggtcttgcatggcctctacaacaaagtcagccagttcccagaactcaccagtcacaccatgcgcttcaacgccttcatcctcggcctgctcaagtgagtgcagaatggcaagatgtaagcagccagccccaccatagctttgcttcctctcaggggctggctgcctggaaggagaccggaggggtgtctgctccttgggatgcctttaccccaccgaggtgcagCCTCTCGGCAGGCGCTGACACCTCCCACCATCAGGCCCCTCCACTTCAGAGGCCATGGCCTGGCCTGTTTAGAAAGAAGCTGGGTTTCTCAGTGTTCGGTGTCTGCAAACGGTGATGTCTCA
This mutation is a 1006 bp deletion beginning at Chromosome 4 position 43,423,415 bp and ending after 43,424,420 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
41624 | GTG TCT GCT CCT TGG GAT G | Wild type Forward | A | |||
41625 | CAT GGC CTC TGA AGT GGA G | Common | A | |||
41626 | GCA TGC TGG TCA GTA GGT TAG | Mutant Forward | A | |||
41627 | Fluorophore-1 | TTA CCC CAC CGA GGT GC | Quencher-1 | WT Probe | ||
41628 | Fluorophore-2 | AGC TAC CTC TCG GCA GGC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
41624 | 0.40 uM |
41625 | 0.40 uM |
41626 | 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 |