Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr17:21557048+21557150 103bp TCTTCCCTCATCTACATCCTGT GGAATGTCAACAGACCCTGA
Mut= 112 bp
Wt= 103 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TTTTTATTTTTTTGGTTTTTTGAGATAGAGTTTCTCTGTGTAGCCCTGGCTATCCTGGAACCCACTCAGTAGACCAGGCTGGCCTCGAACTCAGAAATCCACCTTCCTCTGCCTCCCAAGTGCTGGGATTAAAGGCATGTGCCACCACGCCCGGCTACGTTTTGATTTTTTTTAATTTTTTGATTATTTTGATGATAATAGTCTTCCCTCATCTACATCCTGTCTCCTTATATCCAGAAAAGCATCCTACCCTGTAgcacaccgttgaacaataattttttatttcagggtctgttgacattccgagatgtggctgtggacttctcccaagaggaatgggaatgcctggactctgctcagagggctttgtacattgatgtgatggtagagaattatagcaacctggtctctgtgggtgagaacattttggttgtacaattcttaactcattctttgtaagtactggcccTATAGGCTGTAAACTCTATTAAGCTGTCATGGAAAAAAACAAGCCTAGACAAAGTTCAATAATTAAAAAGAAATTTTCCAAGAGGTTCAGCTGCCAACGCTAGCAGAAGACAGCCAGAGGGTGTGACTACTATGAGCTAGTAAGCATGTGATGGAGACACCGGACACAAAGAGAAGCATAGTGATTCGTGCTCTCGGAAGAAGAACTGGAACTGAAGATGGAAGCTCAGTGAGGAACAGCCTGATGTGAGAGGCCTGTGCTGCCACATGAGGC
This mutation is a 213 bp deletion beginning at Chromosome 17 position 21,557,103 bp and ending after 21,557,315 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48058 | TCT TCC CTC ATC TAC ATC CTG T | Common | A | Common | ||
48060 | GGA ATG TCA ACA GAC CCT GA | Wild type Reverse | A | Wt R | ||
48061 | GAA CTT TGT CTA GGC TTG TTT | Mutant Reverse | A | Mut R | ||
48062 | Fluorophore-1 | CAC ACC GTT GAA CAA TAA TTT TTT A | Quencher-1 | WT Probe | ||
48063 | Fluorophore-2 | CCT ACC CTG TAT ATA GGC TGT AAA CTC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48058 | 0.40 uM |
48060 | 0.40 uM |
48061 | 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 |