Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr11:69001876-69001977 102bp TTGAAAGCAGGTGTCCTTGG TGGCAAGAATCAGTTGGGAAG
Mut= 117 bp
Wt= 102 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CGCATCTCAGTAAGCACTGTGCCATTGAACAGCTTTTCTTTTGTAACATGATGTGGCTAAGTTTCCCATACTGGGTTTCTGTATGGGTCTATGTACCTGAGTGCAGCGCCAATGGAGGCATCGGGGCCCCCTAGAACTAGAGTCTCAGGCAGGTGTGAGCCTGACATGGGTACAAGGAACCAAACTTGGAATTATTTCTCCATCTTCTGATTAATTTTTGAAAGCAGGTGTCCTTGGCATCCAGTCTGAAACCAGAGGCCAGTGgtgtgttgggtcgatagcagtgggcaagatctaccttcccaactgattcttgccaaggccttcagattataaccatctgactctttcctcaggctgaatttctcgactccagcatcgaccaacattgtttctgtgtgccaggcagccgggctgagggctgtggatcgggtagagaccacccgacgttaccgactttcggtaaggtgtgggcattgtggtaggagatcaagaggagcgagtgggccagagattagatcctgaactcgagcagagtcatgggagaagcagggggaaagctcttggcttttggcctgtcctgtatgcttaggaggtttggttgtttgctgcttgaaggtacctgtgaagggagaatcggccagcctgactaaccagacttatctgtgcatccttttagtctgaacaaagacgctGCATGGGTCTGAGTGTGCATAGCTCAGTGGTGGGAATTCACCTCCTATATACAAACCCTGGCCTTGATCTCTGCACTAGAACAGAAAAGTGGTGCAATGTGGTAGTAGTGAGGTGAATAGCTTGGAGGTCCAGAGCGAGTCCTTTTTCTCTTTTCTTCAGCCTTGTCTTTTGTTTTCTTATGACTGGATCTTCCCAACACCAGTTTACCGACCACCCTACAGCTGAAATGGAAGCCATCTCTCTGGCCGCCTTGCACGACCGGATGACAGAGCAGCACTACCCTGACCCCATCCAGAGCTTCT
This mutation is a 431 bp deletion beginning at Chromosome 11 position 69,001,500 bp and ending after 69,001,930 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48017 | TTG AAA GCA GGT GTC CTT GG | Common | A | |||
48018 | TGG CAA GAA TCA GTT GGG AAG | Wild type Reverse | A | |||
48019 | AGA TCA AGG CCA GGG TTT GT | Mutant Reverse | A | |||
48020 | Fluorophore-1 | CGA TAG CAG TGG GCA AGA TC | Quencher-1 | WT Probe | ||
48021 | Fluorophore-2 | CAT AGC TCA GTG GTG GGA ATT C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48017 | 0.40 uM |
48018 | 0.40 uM |
48019 | 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 |