Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr1:56637303-56637408 106bp TGTGAAGTCTGTGTGGGTAAAG CCGGAAGAGGTCTCTAAATCTG
Mut = 119 bp
Wt = 106 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
CCAATATAATCTGCAGAGTACAAAGCTGTTCAGATGTGAAGTCTGTGTGGGTAAAGTGTACAGGATATGgctgaagcaccttcggaaatgcaggctgtttcccctaacagtctatcaacagatttagagacctcttccggcgagccttggtgtgacaacacaggtcagaaggactcagacttatgggctattattgaggaaagtgcatttcaggttttggctcagcgattcttgataaagaggccaccgcataccctctgtgcctcagagccagatgaagacagtaacttattttcaatgaccttccccagaaaactctggaaaatagtgggaagtgacaaatttaagtctatttggtgggatgaggacgggacttacatagtgatcaatgaagagctcttcaagaaggaagtgttggaaagaaaggcccccttcagaatatttgaaactgatagcatgaaaagtttggttcgacaacttaatctgtatggattcaggaaaatgagacagaacttccaaagatctgcttctctccccgactttctggctgaagaaaaaggaatttctgcctcatgtaagttacaattctaccagaatccaaactttctgagagactgtccccatcttatagaaagaatgaaaaggagaattgggattaaaacagtttctcgtggagctgctccttcccttcctgatttcaagaacaaacacttcagaccagacttggtcaagatggatgaccatagtttgggtgtagctgtccaaaccagtgagagaaacttgttgtccagctctagtatttcaaatgtatatctgagacagaagccttctcttgctcaagggggttctgatacgatggatgtcatcagaagcgacttttctcttgcaacgccaagttccttcagaccacctgaagaaattctaacacatcggcctgctccattaaatgaggtgtcctctctttgtatggactcacagagaatcttcactcaagcaaacgacagcactgtgaatttcattataacctctattactcaaaaccgcggggacatgtctcacttgtggaatagttgtattgaaatgcaaggagagccttcctttcagcctgggtttcctcacttttcatccagttcatctacttattctgactcaaaagcaaagggcgaacctgagttaccgatacacaaggaaagagtagcatctaccacgttggcatcaacatcaaaccatcactcatcttcttgagGCCATCAGTTTTATGATTACCAACAATCTACTTGGTTCTTTAGGAAGACATTCATCATAGCAGAGATTAGAATTCATGTTGACAAACACCTGGAGTGTACCTGAGATTATTGTAATAGAACAATAAAACTCTATGTGC
This mutation is a 1177 bp deletion beginning at Chromosome 1 position 56,636,197 bp and ending after 56,637,373 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
49335 | TGT GAA GTC TGT GTG GGT AAA G | Common | A | |||
49336 | CCG GAA GAG GTC TCT AAA TCT G | Wild type Reverse | A | |||
50577 | TGT CAA CAT GAA TTC TAA TCT CTG C | Mutant Reverse | A | |||
50578 | Fluorophore-1 | ACC TTC GGA AAT GCA GGC T | Quencher-1 | WT Probe | ||
50579 | Fluorophore-2 | ATA TGG CCA TCA GTT TTA TGA TTA CCA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
49335 | 0.40 uM |
49336 | 0.40 uM |
50577 | 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 |