Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr18:21086048+21086146 99bp AGCGCAATTCAATGTTGTTG AACAGGGTGTTTGCCTCACT
Mutant= 103 bp
Wild Type = 99 bp
Wt Sequence: ccagagcagccatgttgctgggttgaaattcctttacagagcgcaattcaatgttgttgtgcagtgtgtccccccactGAAAAGGGAAGGACATATAAGGTAAGGAAGTGAGCTCCACAGTGAGGCAAACACCCTGTTCCTGGTCTGTATCAGGTGCTGGTCTTCAGTGGGAAACATTCATGCTGGGAAGCAGGAGTTGTCCATTGGGACAAACTGTGACAGAATAGCAACAGTTCAGCATGAGTTCCTCCACGCCCTGGGATTCTGGCATGAGCAGTCACGTGCTGACCGGGATGATTACGTCATTATAGTTTGGGACAGGATTCAGCCAGGTATGTTTCTCTTACATGCTTAATTACAGGTCAAGTTTCTGAGCAGGTAAGCATCAGTAGCAGCCTGGTAATGGGTGTTTGGAGACCAGATACACTTCATCATGTCTACAAATTCATCTGATATCAAGGTGCAATCAGTTGTCCAATAACAGGGAGCTAAGTAGCCTGGTAtcttctctaaatactctcaacttaatgttttaaaatgattatctttggagctc
Deleted Region: GAAAAGGGAAGGACATATAAGGTAAGGAAGTGAGCTCCACAGTGAGGCAAACACCCTGTTCCTGGTCTGTATCAGGTGCTGGTCTTCAGTGGGAAACATTCATGCTGGGAAGCAGGAGTTGTCCATTGGGACAAACTGTGACAGAATAGCAACAGTTCAGCATGAGTTCCTCCACGCCCTGGGATTCTGGCATGAGCAGTCACGTGCTGACCGGGATGATTACGTCATTATAGTTTGGGACAGGATTCAGCCAGGTATGTTTCTCTTACATGCTTAATTACAGGTCAAGTTTCTGAGCAGGTAAGCATCAGTAGCAGCCTGGTAATGGGTGTTTGGAGACCAGATACACTTCATCATGTCTACAAATTCATCTGATATCAAGGTGCAATCAGTTGTCCAATAACAGGGAGCTAAGTAGCCTGGTA
425 bp deletion beginning at Chromosome 18 positive strand position 21,086,087 bp and ending after 21,086,511 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
37463 | Fluorophore-1 | TGT CCC CCC ACT TCT TCT C | Quencher-1 | MUT Probe | ||
37464 | AGC GCA ATT CAA TGT TGT TG | Common | A | |||
37465 | TGA GTT ATC ATG AGC TCC AAA G | Mutant Reverse | A | |||
37466 | AAC AGG GTG TTT GCC TCA CT | Wild type Reverse | A | |||
37467 | Fluorophore-2 | AGG GAA GGA CAT ATA AGG TAA GGA AG | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
37464 | 0.40 uM |
37465 | 0.40 uM |
37466 | 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 |