Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr14:55632770-55632860 91bp GTGGTGGGAAAGGAATAGCC TGTATACCCCTTCTCTGCCTTC
Mut = 111 bp
Wt = 91 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
GAAAACTAAAGTTTTTTGTGATTTATTTTTCATTTTTTAATTTTTTGGTTTTTCTGAGACAGGGTTTCTCTGTGTGGCCCTGGCAGTCCTGGAGCTTACTCTGTAGACCAGGCTGGCCTTGAACTCAGAAATCCGCCTGCCTCTGCCTCCCAAGTGCTGGGATTAAAGGTGTGTGCCACCACTGCCCAACTAAAACCCTAAAGTTTTTGAGAATGGCTGAAAATGATCAGCTAAACCCAGATAGGGAAAACAAGACTGAATTGAATTCAGCCTGTCCAGTGGTGCTGACCGAGCCAGATCAcaatgcccccctcctttcctcacccccagctgtcggtggtcttatggggtctgccttaggaaagaccgtgcacatacctgcattctctttctggccactccttgggcctggggcattgtttgcaagggaagggtggtaaaaggtgtttctatatgaatggacctcctctttgtaggcattctgtgagtgtcagcttggcccagctctcagccaccatttttcgaaaggaaggcctacaaggatggcctcagttcatgaaccttcttcagcacagcactcatagttcccatagccccgagaaagaggtactggcactgccctgttggcaggaaggagggacagaggctggatagaccacgagaccgtctcttctcccctgctcctgtacaggtgggacttctgctgctgagtgtggtggtgtcttcccagcccgaggcattccatgcccaccagcacgagctgcttcagcttctgaatgagacgctcagtgacgtcagctttcccggagtgctcttctactccctgcgcactctgactgccatcgcccgctatgtcaggccggatgatgtggtaagtaagacgctgcctgtatctctcggtttcgtgccctcgctttcagaagtcttggcccaagctgctttcgagttttattccgcctcccgttcttcctaagggttgagggcatagcgtgttagctgggcctggctggctccttagatggaggctttgaggaagcctccagctcctggcttctgggatgagaccacctgcgctttatgtgattacgctcattcctgtgtcttccagtctcttgcacggatgttggtgcccaaagtcgttacggcactgaggactctgatccccttagatgaggtaaggacagacgggaggggtctccttttggaaggaaaatggaggcacaagcaccatggtcccctagaggagcgagtaccctgtgacttacttgtctgtctgcaggtaaaggcctgtgaggccctggaggccctggatgaaatgctggagacagagttgcccatcatcaacccccacctctctgaggtcctcacgttttgcctggaggtgagcccggggtgggtgctgctcctgctcgctggtttgccgttaagttttcttgcttgtttctgattcttgtcggtgtctgtataggtggctaagaatgtggcccttggcgaaccactccgcgtacgtgttctctgctgcctcactttcttggtcaaagtcaaaagtaaggtacgttccctctggggaagttaccccatcctgctgtctccgccattagttgtgtttgtaggcttagtcctgctcgagccctggcccagccttctctccctgcctaggagacggggagtctctggctggggtccttgtggttgggaggctgggattttcccatgagcctgttctgggaagatgctcaaggctagccctcccctcttaggccttactaaagaaccgcctcgtgcctcccttgctgcacgcccttttccccctcatggctgctgagccacccatgggccaactggatcctgaagatcaagattcggatgacgatgacttggagatcgggctgatgggggagactcccaagcactttgctgtacaggtgatgtattggccttctcgtcactgtcacccaatactcagagaaacagacaccttaaggggaggaagtattttcatggcttcggagatttccatttactgtggtgggaaaggaatagccaaacagtggttcaggcctcggcaaacCAGAACCATTCACGTTATGGATAGAAGGCAGAGAAGGGGTATACAAGGAGGAGACAGGGCAAGATAGCTCCCATAGGATGTGGCCCCAACTAAGCTCTGCCTTCTGCCTTTGGCTCCTCCTAGTGACTGGCATTAGATTAAGAGATTAACTCATCTTGAGGTCAGAGCTCTCCTGATCTAATTGGCTCTGAAAATATCATCACAGTTTGTCTCCTAAGCACTTCCCAGTTCAGTCAAGTGGATTATTAAAATCAGCCATCGCACCCAGCTGACCTGGTGGACACCTGTAAGCTCAGTCCCCAGCCCTTGGGAGGCTGAGGTAGAACTGTAAGCTTTGGGCAGTCTGAGCCAT
This mutation is a 1170 bp deletion beginning at Chromosome 14 position 55,632,815 bp and ending after 55,634,584 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48949 | TGT ATA CCC CTT CTC TGC CTT C | Common | A | |||
48950 | GTG GTG GGA AAG GAA TAG CC | Wild type Forward | A | |||
48951 | CCC AGA TAG GGA AAA CAA GAC | Mutant Forward | A | |||
48952 | Fluorophore-1 | TTC AGG CCT CGG CAA AC | Quencher-1 | WT Probe | ||
48953 | Fluorophore-2 | TGA CCG AGC CAG ATC ACA G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48949 | 0.40 uM |
48950 | 0.40 uM |
48951 | 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 |