Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr15:75919296-75919442 147bp CAACAGAAACGGCAGTGCT GGACAGAAGGACGAGAGCTG
Mut = 153 bp
Wt = 147 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
AACAGAAACGGCAGTGCTATTTAAGTACGATGAATTGACTTGATCAGccccaaggacaagcaaccctaaggagccttaggctcacactgggctgggcaagctgtgtcctatcttcccagtccttaccagctctcgtccttctgtccaggcaaagtagaagctaaacaagtgctggccagtgcaccaacggacaacaacctctgccacttcagggtgagtgggacctagagggccttgtctttctgaagcatttatcaggggcaggtatggcagacggtttgagatggtttggtctgtccaggggaactaggcgtcagaggccaggaagaaagttctggaaggactagcatagatgcagtgtgtgtcttatgtagggtgggtgtggacactcattaggagggaaggttctagaaggcacaacttaagagtttgaggagcagggcctgttgggtttgtcgtaggcgttggagagtacagcaagtacagacattggtgacccACGGGATACGGAATGGTCGATGGCACGCAGGTCAGCCTGTGTAGGATGCTGCCTCACACAGCCTCATTTCTGGCCCTTGTGTCTCTTTAGGCTCTGGGTTGCCAGACTACTCACTCCAACCATGAGGTGCTGCAAAACTGCCCACTTGTCATCTTTGCCACCAAACCCCAAGTCCTGCCAACTGTCCTGGCGGAAGTGGCCCC
This mutation is a 452 bp deletion beginning at Chromosome 15 position 75,918,943 bp and ending after 75,919,394 bp (GRCm38/mm10). There is a 2 bp insertion (AA) at the deletion site.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
51315 | CAA CAG AAA CGG CAG TGC T | Common | A | |||
51316 | GGA CAG AAG GAC GAG AGC TG | Wild type Reverse | A | |||
51317 | GGC AAC CCA GAG CCT AAA GA | Mutant Reverse | A | |||
51318 | Fluorophore-1 | CTG GGC TGG GCA AGC TG | Quencher-1 | WT Probe | ||
51319 | Fluorophore-2 | AAA CGG GAT ACG GAA TGG TCG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
51315 | 0.40 uM |
51316 | 0.40 uM |
51317 | 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 |