Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr1:161031484-161031574 91bp TCAGTGAATCAGAATAGCATCG AGACCCCTGACAATCAGCTC
Mut= 91 bp
Wt= 91 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
AATACCTTGTACCAGGCTCGGCAGCTTACAGTAATCCTAGCACTTAGGAGGTAGGATCAGGAGTTTGAGGCCAGCCTTATTACTAACAAAACACCTGCTGGCAATTCTGTTTCTACATCCAAACATAAGGAAGATCTCTCTTTCTATATGAAGAATTCTTTTTTTCTCCCTGAGACAAGATTTCACTTATTTAGCCCATACTCATGATTCTCCTGCCTCTTCGGTTTTCCAAGCGCTAAAATTACGTATGTGTAGCTGGCACTCATGGAAAGAttttccatttgtagggatgtttgaactaaaaagcacaaagggagtcaaactagtactaagactgtgctttgctctttcagcagggttgaatgtctgcctgtgctttcaggcatgaccatggagaaaagtgggaacatacaattggagatccccgacttcagcaactctgttctgagccatctcaaccagctacgaatgcaaggccggctctgtgatattgtggtcaatgtgcaaggacaagcttttcgggctcacaaagtagtactggctgccagctccccttatttccgggatcacatgtccttgaatgaaatgagtactgtgtccatttctgtcatcaagaaccctactgtttttgagcaacttctttctttctgttacacagggcggatatgcctgcaactagcagatatcatcagctacctaacagctgctagttttctgcagatgcagcatattatagacaaatgtacgcagatcctagagggcattcacttcaaaattaatgtggctgaggttgaagcagaattaagtcaaacaaggacaaagcatcaagagagacctccagagtctcatagggctacaccaaatctaaaccgctcacttagtccacggcataatgcctcaaagggaaactggagagggcaggtcagtgccgtgctggatatccgagagctcagtcccccggaggagcccaccagtcctcagataattgaacagagttctgatgcagaaggccgggagcccattcttcgaatcaacagagcaggacagtggtatgtggaaacaggactagcagaccagggggcccggagtggtgacgaagttagggttcttggagcagtgcacatcaaaactgaaaatctagaagagtggcttgggccagagaatcagccttctggagaagatgggagtagtgcagaggaagtcacagccatggtgattgacaccacaggccatggttctataggacaggaaagttatactttagggtcttcaggagccaaggtagctcggccaaccagcagtgaagttgacaggtaagttttgtttctccttttaaagctattgtacagacatcattaaataacctttctgtgttacagagggcatctttttttacctgatattcttattcagcttactttgggagactggaagtatgtcagaagacttgcattttcagcacaggaggctcctggatctgaaatattgacgatgttatgagaactgtggtaaactgtggtaaactgtggtaaaattagccacttcagtgaatcagaatagcatcggcagccactctaggatcagtaattgccagggctctcTGCACTAGGCAGGGAGCTGATTGTCAGGGGTCTGGTAACATTGCTGTTTCTCACACAGGTAGGTAACCTTGCGGAGTCTGACTTGCCTTATAGCACTGTGTATTTCTCTTTGCCCTTACCGACAGAGACAATTTGGTACCATCAATAATTTTCAAATTAGAGTAATTACACTACAGTAATTCTAGAGAAAAGCTTTTGAATGTTTTCCAAGTGTCACCTGGTAATGTTTCCTTTGAAGCAGCTACTACCCCAGGCCTGTTTCTTCCTAACATACTGAGTCTG
This mutation is a 1328 bp deletion beginning at Chromosome 1 position 161,031,517 bp and ending after 161,032,844 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48635 | TCA GTG AAT CAG AAT AGC ATC G | Wild type Forward | A | |||
48636 | AGA CCC CTG ACA ATC AGC TC | Common | A | |||
48637 | CCT CTT CGG TTT TCC AAG C | Mutant Forward | A | |||
48638 | Fluorophore-1 | CTA GGA TCA GTA ATT GCC AGG G | Quencher-1 | WT Probe | ||
48639 | Fluorophore-2 | CGT ATG TGT AGC TGG CAC TCA T | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48635 | 0.40 uM |
48636 | 0.40 uM |
48637 | 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 |