Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr6:138151016+138151144 129bp TCCACATGGGGTTACAGTGA AATGCCTGTGCTCTGTCTCC
Mut = 131 bp
Wt = 129 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
TAAATAGGTAAATACCCCTACTGCTTTGTCATATCTCACAATATTTGGAAGTAGGGCGCTCATTTTGGTTTGTTTATGTTGTTACGTCGATGCTAGttgagcctggggccttgagtgctgtcatcacgctatccccagcacaagtgaattttttggttgttcttctttttcgtatttggaataggtttttgccaacccagaagactgcgctggctttggcaagggagagaatgccaagaagtttgttcgcactgacgagaaggtggaacgcgtgcgcaggtaaaccagtgtctctggaaattcctcactcaagaatcatgctcatggagtccagccaggatctcaggatcttagagactgggggaggggggatgatccacttaatctctgtagtaggtgtaacaactccagcaaattagatacatagagttctccacatggggttacagtgactcttgtcagcaattttgttgcagttctagactttattggggtcagtggctgtactctgtttatagatacataaagataatagtatgcaggagacagagcacaggcattagATTCTTGCAGCTATGTTATTTTTCTCTCAAATTCAGATGGTCAGTTCACATAAAACATCAAAGATC
This mutation is a 467 bp deletion beginning at Chromosome 6 position 138,150,680 bp and ending after 138,151,146 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
49668 | CCC CTA CTG CTT TGT CAT ATC T | Mutant Forward | A | |||
51476 | TCC ACA TGG GGT TAC AGT GA | Wild type Forward | A | |||
51477 | AAT GCC TGT GCT CTG TCT CC | Wild type Reverse | A | |||
51478 | GTG AAC TGA CCA TCT GAA TTT G | Mutant Reverse | A | |||
51479 | Fluorophore-1 | ATT GGG GTC AGT GGC TGT ACT CT | Quencher-1 | WT Probe | ||
51480 | Fluorophore-2 | ATT TGG AAG TAG GGC GCT CAT TT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
49668 | 0.40 uM |
51476 | 0.40 uM |
51477 | 0.40 uM |
51478 | 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 |