Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr1:33747215-33747334 120bp TTCTGATGAATATGCAGTGTCC TGTGTCCACAGAAGGCAGAG
Mutant= 134 bp
Wild Type = 120 bp
Wt Sequence (deletions in lower case):
GGTGGTATATTCTGATGAATATGCAGTGTCCCCGCCCCCGCCGTGAGCCCTCCTCTGGCTAACctctgctcgctctgaagttgcttggctgcatcccagctgcccatgtctctgccttctgtggacacatagactcaggatgatctctggccctaactgtgtgaagacttttcctcagttatgctgatatgccgtagtacttttggcttttacctttagaaaaatttacagaagtgcttacagaaactaaagatagcgcaagtgcatgctgtggcgttgctgacgtgggaagtttctgcctcatactgactgatgtttcgtgtttaggagaaagagaggaactaaacctgactgccaaccgtctgatgggccggaccctcacggttgaggtctcggtggaaacgatccgaaacccccagcaggaggaatccttgaagcatgccacgaggattatagacgaggtggtcagcaagttcctagatgacctggggaatgccaagagccacttaatgtcactttacagtgcctgctcatcggaggtgccgcctgggccggtggaccagaagtttcaatcgatagtcatcggttgcgctcttgaggatcagaagaaaatcaagaggcgattggagactctgctgaggaacattgacaactccgacaaggccattaaactcctagagcatgctaaaggagctggttccaaaagcctgcagaacactgacggcaaatttaattagccttcaaattcaccggcccttacaaaagatgtagaaaagaaaactactattttcaatagctaggtctctatgttaggcataaccacttaattgatattggtaattatttcagatggggaagtattccagtgttggcagacacaggtaacacttaacactaggaatatttcagtcacctactttgaagttttggacagtgttcccatcttgaactggactctagcactgtatatttaacttctgaagatgcacatgtagtgtgtgtgtgtgtggggggagtaaaaaaagagggggagcagcaaaagtcccactgccctttccttgacttcagcacagagcatccagcctctggagcagtgcactcacatcgcagtgtgagcagagcacccagccctggagtggtgcattcacaccacagtgaacagaacacccagccctggagcagtgcactcacaccgcagtgtgaacagaacacccagcccctggaacagtgtactcacagcacagtttgaacagagcacccagcccctagagcagtgtattcacacctcggtgtgaagagaacacccagcccccggagaggtgcactcacaccagtgtgaacagatcactcagcccttgagcagtgcactcacagcacacagtgtacctagaaggataagcatatcagaacttcaccctgcatggtctccatgtttcaggcatctgttttctctatgcatatgcatctcataccatgaatttttaggtgtgcgtggaagcatgacaaatgtgaggagcccaccttcattttaccactgtgaagctccgcccccaccccagggccagtgctgaccgctctcttcatgtgctgccttgagctcacgcgtgtgcggcgggagcacttgcctaaccacttccctgtgggcagttggatggtttcaatttgttgctgtgactacaacatacttgtatctgctgagctttattctgttcttggtcttttttatataaaattccattaatgggttcatcaggccaaaggatatatacattttcatggtctagaaacatatttctaaatttttataagttatatcagtttgtactgtcttcagtaattaaaagaatccatttcgctgaacttctagcattggccattatttttctaccgtcagacacaaagccttctatttaatgggtaccttgtttcctaagaaggctggccagcgtggtcatcttCCCGAGTTCTTTCTTTGTTTTTTGTTTTTCGAGACAGGGTTTCTCTGTATAGCCCGGGCTATCCTGGAA
This is a 1895 bp deletion beginning at Chromosome 1 position 33,745,386 bp and ending after 33,747,280 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46693 | TTC TGA TGA ATA TGC AGT GTC C | Common | A | |||
46694 | TGT GTC CAC AGA AGG CAG AG | Wild type Reverse | A | |||
46695 | TAC AGA GTG AGT TCC AGG ATA GC | Mutant Reverse | A | |||
46696 | Fluorophore-1 | CTC GCT CTG AAG TTG CTT GG | Quencher-1 | WT Probe | ||
46697 | Fluorophore-2 | CCT CTG GCT AAC CCC GAG T | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
46693 | 0.40 uM |
46694 | 0.40 uM |
46695 | 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 |