Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mut= 158 bp
Wt= 166 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CAGGCAGAGATAGCTTGTGCAAAGCCCGGGGGCAGGAGCCTTCCTGTATCCGTCTGCGTTCCTGGTATGGAGGCTGTCGAAAGATTCCTTTGTTACTCTGGCTGTAGAAGAGAGACTGGTGGGAGAGAGTCTCTGCAAAGCAACTCAGTGGTGGGCACAGACAGggcgtgtgcctggggttaatgacataactattttggaaaatgagctttctatccaggaaaggggctccccatatgttagtttgcttgcttgtttctgaaggaaagagcccggggcacaatcagattgtgtgccaatcgttttgacatgcctcttgctcagaagggatgcctggccttgcttctgtcccaggaaggcatgctgacagatgcggagagagtaattctcccaccccatctctttcccctctccctcatcctctccctctccctccctcacaggtgctttcagaacttagagtggttattcatatatcaaaacctgctcaaagtaagttaccctatacttggaatgtcagaggactgcttatacctcaacatttatgcaccatgccatgccaataacggctccagtctcccggtaaggctgcctgctgccctggggttcattagcaggtatgggtaacagctgggcatggggcatctggagttagacctagcctcccattgggtaagaggaaaagtccatcacacaagtaacttgtttgttggttggttttttttcttttctttccctcgagacagggtttctctgtatagctctggctgtcctggaactcactttgtagagcagGCTGGCCTCGAACTCAGAAATCTGCCTGCCTCTGCCTCCCAAGTGCTGGGATTAAAGGTGTGCACCACCATTGCCAGGCACACAAGTAACTTTTAAAAACATATTATAATGGAAGCTGTCCTCTGTCATTGTCTCTGTTGAATATTTCG
This mutation is a 631 bp deletion beginning at Chromosome 8 position 93,530,546 bp and ending after 93,531,176 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
45562 | AAC TCA GTG GTG GGC ACA GA | Common | A | |||
45563 | CAA AAC GAT TGG CAC ACA | Wild type Reverse | A | |||
45564 | GAG ACA ATG ACA GAG GAC AGC | Mutant Reverse | A | |||
45565 | Fluorophore-1 | AGG AAA GAG CCC GGG G | Quencher-1 | WT Probe | ||
45566 | Fluorophore-2 | CAC CAT TGC CAG GCA CA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
45562 | 0.40 uM |
45563 | 0.40 uM |
45564 | 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 |