Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 132 bp
Wild Type = 133 bp
>chr2:125773330-125773462 133bp AGCCCAGCCCAAGTCCTA GGATGATAGAAGCCCATAACTTG
TGGGAAACAATAAATGGATGTGGAGTGTGGCAAGCTCTCAGGCTGGATTCTCTTACGATGCTGTGCTCATGGATATTACAAGTGCAGGttatcagcaaaaaaagctgatggtgtcagtggtgcctctccctaggagtattgtgaaggcactgctctctgtcttattgtttgttctgctttgatattactcttggatgtcaatagatttttttctctctcttgtttggttttgcagacaatttcctttatataacaacgatatacgatggcagcagcccagcccaagtcctaccggaccctaccttgcctaccccattatatctgctcagccacctgtttccacagagtacacctattaccagctgatgccagcaccctgtgcccaagttatgggcttctatcatccttttcctaccccttactccagcaccttccaggctgcaaacactgtcaatgccataagcacagaatgcaccgagcgtccaaatcagctggggcaggccttcccgctgtccagtcatcggagcaggaatggtaacagagggccagtggtgccaaaagtgagtggccacactgtgtgtgtagtttttaaatatgctatttctgttgttagtactagtataggtaatttactacttggaccattggccTTTAATGCTTAATTTGTCACAAAGTATTCATCACTAATAAAAAGTGTTGTAGGTATATTATTTAAAAATTTTTTTTCTTGTGATTTATTACCCCTCTTGTTTGAAAGTGTAATTTCAAGTCATCCTCATGATCATTCAGAGAGTTTTACATAAACATAATTTACAATTATTTCTAAATTATTTTGATTTAGTGCTAAAA
This mutation is a 562 bp deletion beginning at Chromosome 2 position 125,773,086 bp and ending after 125,773,647 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44462 | AGC CCA GCC CAA GTC CTA | Wild type Forward | A | |||
44463 | GGA TGA TAG AAG CCC ATA ACT TG | Wild type Reverse | A | |||
44464 | Fluorophore-1 | CTC AGC CAC CTG TTT CCA CAG | Quencher-1 | WT Probe | ||
44465 | ACA AGA GGG GTA ATA AAT CAC AAG | Mutant Reverse | A | |||
44466 | CGA TGC TGT GCT CAT GGA | Mutant Forward | A | |||
44467 | Fluorophore-2 | AGT GCA GGT TTA ATG CTT AAT TTG TCA C | Quencher-2 |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44462 | 0.40 uM |
44463 | 0.40 uM |
44465 | 0.40 uM |
44466 | 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 |