Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr14:32863698+32863857 160bp TGGTGAAGATGACGAAGCTC TTGCAGACATACTGCCCTTG
Mut= 160 bp
Wt= 160 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
ACTTTTGAGAATGTGTCTGTTCAGATGGCCAGTGTTTGGCATGCCATGTGAGTGAGACCATCGCATATAGGAATGTGGAAGCCATAAGGCCCATAGAAGGGATTCTTAGTGGCTTCTTTGGACTCAGGATCTATGGTCTGAACTGAAGCATACAAACAAGCTAAAGTAAAATAAGAcgaaggaaggagactgaaactagaagggatatggggctctcccagtaccccaaaagatgcttcagaattccaggtgtttcctggtcatggtgaggctgggaagtatagagagtgccaagaccactgcctctgccccttcagagcatccagacgccaatagcatgtggcttgaccctggcttctgggaatggcctggctgcacaaacccaggctgcggccttcattcacctttccatcctcttccattgcagaagtctgtggtgctctcaatgtcacggtgtccccaggacctgtggtggactacttggaaggggaaaatgccaccctcctctgccacgtctcccagaagagacggaaggacagcttgctggccgtgcgctggttcttcgcccctgacggctcccaggaggccttgatggtgaagatgacgaagctccggataattcagtactatgggaacttcagccggactgccaaccagcagaggctacgcctgctcgaggagcgtcgaggggtgctgtacaggctgtctgtcctgacgctccggcccacagatcaagggcagtatgtctgcaaagtgcaagaaatcagcaagcaccgcaacaagtggacagcctggtccaatggctcctcggcgacggaaatgagaggtgagaacgctgttggcatctcggaggaggcgcagtgtgtggtagaaggtggtcctggcgttagtcagtatgtaaatgctggtcgcctcattggctctgctagtatagttagtccacgtcttgtggcctctgtgggccaaatgcctgtgagtcaagttatgaggatgaggacagctcttggacactggctgatgtggaaggtagatatggaggcagggacaacagagtgagtgactacctccgtattagtCATGTCCTGCCTGAAATATTGAACTTTGTATGCTGTCAGATAAAGAAATCTAGCCCCTACTTACTTTGGACAGAGGCGTGTGAGGGTATGGCCTGAGCTGTGCTTACTGTGGACTTGGACTGGGACTTTGTGGTTC
This mutation is a 900 bp deletion beginning at Chromosome 14 position 32,863,282 bp and ending after 32,864,181 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46085 | TGG TGA AGA TGA CGA AGC TC | Wild type Forward | A | |||
46086 | TTG CAG ACA TAC TGC CCT TG | Wild type Reverse | A | |||
46087 | ATG CCA TGT GAG TGA GAC CA | Mutant Forward | A | |||
46088 | GTT CAA TAT TTC AGG CAG GAC A | Mutant Reverse | A | |||
46089 | Fluorophore-1 | CAG TAC TAT GGG AAC TTC AGC CG | Quencher-1 | WT Probe | ||
46090 | Fluorophore-2 | TGG AAG CCA TAA GGC CCA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
46085 | 0.40 uM |
46086 | 0.40 uM |
46087 | 0.40 uM |
46088 | 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 |