Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr10:39894595-39894685 91bp TAGCGTGCTGGCATCCACT TCAGGTGGCCAACACTCAC
Mutant= 103 bp
Wild Type = 91 bp
Wt Sequence: AAAGGTTATGACTAGAAGGCCACTGGGGAGTCTGTGGCTTTACTGCTGATAATGTGCATGTTTTCTTAGTTCCACTGTGGTAGACTGTCATCTCCCTCCtgtggttttgttacagtgggaaagctactttttggtcagtgttccagcgatagtaagaggctctcttgcttttaactatagggactaagtgttgctatcctggggcccacctttccagacttggcgaagaacgtgaacagaaacatcagcagcctgtctctgattttcgtgggccgggcctcaggatatttatgtggctctatgattggaggagtcctgtttggctgtataaatcattttttacttttgggtaagtaaacaccgctcttctttgtttctgtcaacaaatttacaactcagctctaaatggggaagatggtggatacctgagccaggtagcgtgctggcatccactgtgggagctgggaagtctggactgattccctggGAACTCGATGGGCCCTGGGTGTGAGTGTTGGCCACCTGAGCAATGCTGACTTTACTGTGATATAGGCTCCACTCCAGGCACAGGCACTGGCTGACCCTGAAGCTCTGCTTATCTGGGGTAGGGGCGGGGCTTCTGGGGCCACGGGGGAGGAGGA
Deleted Sequence: tgtggttttgttacagtgggaaagctactttttggtcagtgttccagcgatagtaagaggctctcttgcttttaactatagggactaagtgttgctatcctggggcccacctttccagacttggcgaagaacgtgaacagaaacatcagcagcctgtctctgattttcgtgggccgggcctcaggatatttatgtggctctatgattggaggagtcctgtttggctgtataaatcattttttacttttgggtaagtaaacaccgctcttctttgtttctgtcaacaaatttacaactcagctctaaatggggaagatggtggatacctgagccaggtagcgtgctggcatccactgtgggagctgggaagtctggactgattccctgg
388 bp deletion beginning at Chromosome 10 negative strand position 39,895,021 bp and ending after 39,894,634 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
37468 | TAG CGT GCT GGC ATC CAC T | Wild type Forward | A | |||
37469 | TCA GGT GGC CAA CAC TCA C | Common | A | |||
37470 | GGC TTT ACT GCT GAT AAT GTG C | Mutant Forward | A | |||
37471 | Fluorophore-1 | AGC TGG GAA GTC TGG ACT GAT | Quencher-1 | WT Probe | ||
37472 | Fluorophore-2 | TGG TAG ACT GTC ATC TCC CTC C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
37468 | 0.40 uM |
37469 | 0.40 uM |
37470 | 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 |