Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr10:89772129+89772239 111bp TTCTTTGCGTGGTGACTGAC AGAGAGAAGAGACAATGCTATGC
Mut= 119 bp
Wt= 111 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletions in lower case):
ACAAGTGGTGCTTCTAGAGTTAAGTCAGAGTTTTAAAAAGTTACCCAGAGAATATCAAAATCAACTTCTAAATGGGATTGTGTAATTTCCTTCCTCAGAAAAGTTAAAAACACTATACGTCAGAATTTATTTCTGAAGTGTTTTGGGTTGGTTTCTAAGGCTGGGAGTAGCAGGTGATCCTCAGAGGAGTGGTATGACCTCTGACCCTTGTTCTTTGCGTGGTGACTGACATCTGTGCCATGTGAGCTGCTGTTAGagggtcctctcaatttccctacctttgtgtgcttggttaatagcatagcattgtctcttctctctgatagggtctacagtgaacaaaatgtggtagggactgggcttgcattgtgcctggaaagcagtggtggatacacagttttcatccatttgagggagtcaggaccagacttcagctgtttacaggaatgatgtacacatcggggcccccaaactgtagactcactattagagagttgtggcctggagagcagagaacagcagtgacagacactggacatgggttaaaataaactcatctctgtggtggagtttagtctgctgatgtgtttctagaactctgtatttgctaggttgtctgtagggtgcattagctgacactaaagtaatgtaagtgtggtctctttcagattcactaaaaacttatctcctgacaaaataaacctcagcacccttaaaggagagggggagctgaagaacctggagctggacgaggaggtgctgcagaacatgctggacctgcccacctggctggccatcagcaaggtgttctgcaataaagcctccatcagggtaagcaaggcctggctgtctgcggaggctgcctgctctcacaatagatactcggcttcaatagatACTCGGCTTCAGAACTGCTTATAAAGAAACACTGTGTTCCTAGTATTTACACAGAAAATAGCTGCATGAATGGTTTTCATCCCTTATGAGTATAACTGAAGTAACATATTAACAACTTACATTTACATTTTGTTTAAAACATCTTTATTGATAGTTTAATAAAATTGATTTATTATAAATTAATTGCTTTACTTTGGCATATATTCAATGATAGCCCGCCCCAGGTTTTATTGTCAAATGAAATGTTTTAAATTTTTAATACTT
This mutation is a 641 bp deletion beginning at Chromosome 10 position 89,772,175 bp and ending after 89,772,815 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48394 | TTC TTT GCG TGG TGA CTG AC | Common | A | |||
48395 | AGA GAG AAG AGA CAA TGC TAT GC | Wild type Reverse | A | |||
48396 | CCA TTC ATG CAG CTA TTT TCT GTG | Mutant Reverse | A | |||
48398 | Fluorophore-1 | TAG ACT CGG CTT CAG AAC TGC T | Quencher-1 | MUT Probe | ||
48405 | Fluorophore-2 | TCC TCT CAA TTT CCC TAC CTT TG | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48394 | 0.40 uM |
48395 | 0.40 uM |
48396 | 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 |