Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr5:23776133-23776223 91bp CCCTAAATGTTGATGGAC TGGAGCACATGCACTTCTATAC
Mut= 90 bp
Wt= 91 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TCAAAACCTGGGACTCACCAGTGCTTATATAGACTGGCAGGCCTGAGGCGCCAGGATCCTCTCCTCTCTGCCTCCCCAGCATAACCCAGGCCTGTGTTACCACACCTGACTTTTTCACCTCAAACGCAAGTCCTCATCCCTGCTTGCTCCTCAAGCACTGTACTGACGGGGCCCTAAATGTTGATGGACTTACTTTTGCGTTGGTTGatatgcgctatggtaatattttagaagctcctagtatagaagtgcatgtgctccattaaaataagagaagattgcttggtattatttgccaataaagattttccttaaatctcttgttttgaacaatggtgaactttgtttactttttactaatagtaaaattaggtagaaagtaaaacaacatccacttcttgaaagctttggttttcttttcatcagatactctgactttgtcgttcacgaaatcgggaaagatggacggatcagccatttggatgacttatctgttccggtggatgaagaggtaaaaatgtctctagatcaagctctcttctgtatgtagtcactcttcctaatgatcctgGACAGGACGTGTTACTGTCTGTGTGTGACTCTCTTGCTTTCTTGTGGTTGAAGGACCCTCCAGAAGACGCTCTCACTGTCTTGACAGCTGAGGACAGGCAGCAGTTGGAGGAGCTCCAGCTTTTCAAAAATAAAGAAACCAGTGTTGCCATCGAGGTAAGCGGCGCAGGAAGTGACGTCTCCGGGCTGGGCTGCTTCTCATTGGGTG
This mutation is a 364 bp deletion beginning at Chromosome 5 position 23,775,824 bp and ending after 23,776,187 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
47706 | CCC TAA ATG TTG ATG GAC | Common | A | |||
47707 | TGG AGC ACA TGC ACT TCT ATA C | Wild type Reverse | A | |||
47708 | CCT TCA ACC ACA AGA AAG CA | Mutant Reverse | A | |||
47709 | Fluorophore-1 | ATG CGC TAT GGT AAT ATT TTA GAA GC | Quencher-1 | WT Probe | ||
47710 | Fluorophore-2 | TGG TTG GAC AGG ACG TGT TAC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
47706 | 0.40 uM |
47707 | 0.40 uM |
47708 | 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 |