Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr2:20890030-20890127 98bp TTATTTGGTCTGGGTGTGGA TGTCGAAACTCCCAACACTG
Mut= 89 bp
Wt= 98 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GTGTCTAGAATTCTCAGAACATTGCTATATTTCATGAGCAGTTTGATGTTTTAAAAGGAAGATTTATTTTTGTAACAGGTACTTTAAAAATGTGCAGGCATATTTTAAAAAATACGGGCTTATTTGGTCTGGGTGTGGAGCTCAGACAGCaacgggtttgatcccagcaccatggaaacagagtgaagaggagggagcagtgttgggagtttcgacacagttactcagagtgagtagttctttcaaggagttcctaatatgttgttctccccccccccccctcttctttgaaggtgacagaattataaaagtcaatggagaaagtgtcataggaaagacctattcccaagtaattgctctcattcagaacaggtaagagtcttgaatcctgattaatatgggctaaaaccatatagcacaggtagcttttatacagttctgtgctcctaggacaagggtaacacagaatttcctccaaaactgcaagttgtgtatggtattgccaaatgttagcccactgattgttgagtgggtttgcaagttgtaatattcattgtaatgtttacaatgcaaaatcctatgGGTTCTGAGAGCACTCATTATAGATTTAAAATATGAGTTTTCCACTTTGATTTCAAGTGATAGGTATATAATTTGTTTAAATATAGAAATAAATGCACACTTGGTTTTCTAAAACAAGTCTTACTGACATTCATATTT
This mutation is a 432 bp deletion beginning at Chromosome 2 position 20,889,665 bp and ending after 20,890,096 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
39549 | TTA TTT GGT CTG GGT GTG GA | Common | A | |||
39550 | TGT CGA AAC TCC CAA CAC TG | Wild type Reverse | A | |||
39551 | ACT TGA AAT CAA AGT GGA AAA CTC | Mutant Reverse | A | |||
39552 | Fluorophore-1 | TGA TCC CAG CAC CAT GG | Quencher-1 | WT Probe | ||
39553 | Fluorophore-2 | ACA GCG GTT CTG AGA GCA CT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
39549 | 0.40 uM |
39550 | 0.40 uM |
39551 | 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 |