Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr11:23517366-23517490 125bp CCAACATAAAGTGATGAGGATGTC AAGTGCATTAGGACCCTGGA
Mut= 122 bp
Wt= 125 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GACACACAGATCTACACACAGTCATACACACATATACAACACACACAGCATTATACACACAGTCATACACACAGTCACACACACAGAGTTACACACATACAAAGTTTCACACACACACACACCAACATAAAGTGATGAGGATGTCCTAGCCattattatttgtggtaatgtcgactaagatgtatggatttaaaaaaaaaaaaaaaaaaaaaactcttgatgattttccagggtcctaatgcactttttaaacatgggcttgccgatgaagcctagcattcttgtgcaaaagcagagtaaggaagagatggccacatccttaggagacaacatcaaggcagaagactaccaaaagaaacagacatatgagcctgtgaatgctagagaaaccaaccatgaggtgacatgttctctttcaggggttgctcagggtttaagtttgatcggggaggagagtcgccatgtgggcgtaattagatcataatggttccatgaggcactgtcagtctgcaggcttccactgttagctttcattcttacATTCCTGTTCATCTTACTATTTCTCCTTTGTTGTTTTTCTTATACTCCTGGGATCTTAGATCTATAAAAGTTCTTGCCACTTATCTAGGGCCTAAACTTTTGTCCCATGGAAAACTAAAGTGACTTGTGACCTTCCTATGGCCGCTAAACAAGCACTGTGTCCTTA
This mutation is a 389 bp deletion beginning at Chromosome 11 position 23,517,072 bp and ending after 23,517,460 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
45537 | CCA ACA TAA AGT GAT GAG GAT GTC | Common | A | |||
45538 | AAG TGC ATT AGG ACC CTG GA | Wild type Reverse | A | |||
45539 | GGC CCT AGA TAA GTG GCA AG | Mutant Reverse | A | |||
45540 | Fluorophore-1 | TGG TAA TGT CGA CTA AGA TGT ATG GA | Quencher-1 | WT Probe | ||
45541 | Fluorophore-2 | TTT GTT GTT TTT CTT ATA CTC CTG GG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
45537 | 0.40 uM |
45538 | 0.40 uM |
45539 | 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 |