Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr18:80912681-80912768 88bp TGATGTCCCCAGCGTTTG AAATGGGGTGGGATGAAACT
Mut= 97 bp
Wt= 88 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GGGTGTGGGAGGAGGGATTTCTAAGGCTGGACCTGGGAGGAAGAGGACTGTGGGGCTGTTGTGATGGAGATGTAAAGTGAATAAAAAATAAATTATTGGGAAAAGAAGAATGGTGACTTTGTAATAGAATGTTCAAAGGCATAGGGTAATGATAGGTTAGCATTAGACAGAAAGATTAATGTCcaaggaatatagacaatcaatgttgattgctaactatgtagacttaggttagaccttttaaaaagcctatacaacctatacaatatatgtttatcttttgttttatgcagttgctgtgattggctaataaatgtttgtcaaaggaagaaagagctaaaagctcgaacagtatggcttggatgtccagagaagtgtgaagagaaacatcctaggaattctatcaaaaatcagaaatacaatgtatttacctttatacctggggtaagatgatcgcatcttcaatgtcagttgatgtccccagcgtttgccttctgatgacacatgtagaaactctgcttgttgataacactgtttggtagtttcatcccaccccatttcaaatactttatattttcttggttctgtctacttaagaaaatgcatacttttattattgctttcagttcatgataaacattcatgttataagaacataaaagtatggaaatagtaaagaacaggtaaacatattttagttctggggacatttgatatttgtttggagggaaaattagaataaatgttgaagcctgccaaggaatttcacaaaggaagtatacgatttgttggtcttcatgtgggctctctaacaagtggattgcatggctgggcctcagtaggagaggctatgcctactcctgctgggactagatgtcccagggtgggatggtacctaaggggggtggggtgggggcggtgctcccccttCTGTGAGAAGGGGAGGGGGGCGTGGGAGGAGGGATTTCTAAGGCTGGAACAAGATATATCTCTGTATTTATTTATAGACACCTTTTTATATCAAGCTGTTAATCTCATGCCTTGTCACATTGTTAACTCATTAGTTACTTGGAATACTCAAGTTTAAATATATTAATTAATTATTGTTTTTACTACTTTAAATTATATTATAAAACTAGGTTTATCATTATTTGGAATATTA
This mutation is a 748 bp deletion beginning at Chromosome 18 position 80,912,310 bp and ending after 80,913,057 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
38886 | TGA TGT CCC CAG CGT TTG | Wild type Forward | A | |||
38887 | AAA TGG GGT GGG ATG AAA CT | Wild type Reverse | A | |||
38888 | GGC ATA GGG TAA TGA TAG GTT AGC A | Mutant Forward | A | |||
38889 | TTG TTC CAG CCT TAG AAA TCC | Mutant Reverse | A | |||
38890 | Fluorophore-1 | TGC TTG TTG ATA ACA CTG TTT GG | Quencher-1 | WT Probe | ||
38891 | Fluorophore-2 | AAT GTC TGT GAG AAG GGG AGG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
38886 | 0.40 uM |
38887 | 0.40 uM |
38888 | 0.40 uM |
38889 | 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 |