Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr10:4514262+4514373 112bp GGAGAACCAGGAACTAATGAAGA GGCTGAGCTCTGAAGCTAGG
Mut= 119 bp
Wt= 112 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CAAATTCTCACACACATTATATACAATAATTACTCTTTTCAATTTGATAATGCAGATCCAAATCCAAGAAAGAGAAAATGAAATTGGACGAGGTCAGGAATAAAATGGAGAAATTTTAAATTACAGTATTGACTCTtttgtaggcccttccaacagcatacacattgttaaatccctttgttctcgcccaccccaacctctctgcactttagcttcgagaactccgctccaagatgctttctaaagaagcttgctgtcaggagctgaaggtggaaatggagagctataaagaaagcagtgccagaaagtcagctctcctcacgtctctgaggggcaggatggaggagctccaggaggaatcagcagcggtttcagcttctaaaagcagagcagaggcctctgttcacgccatgatgaaggagaaccaggaactaatgaagaaagttctagaactggatgagaaattacagtaagaaatttggaaacatcccccagTTTGTTAGTGGCTATCCTAGCTTCAGAGCTCAGCCTACATCTTGAGTGATCTTTGAATGTTTTATATGACTTTCCCTTTAAACTCAAGCTGAAAAAACAATTATAGCCCTCAAATATCTTTGTAGTCTTACAAACTTTGCTCAAGTTCAAGTGTTATTAAGTATTTCATCAAACACAGTG
This mutation is a 359 bp deletion beginning at Chromosome 10 position 4,513,980 bp and ending after 4,514,338 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
40302 | GGA GAA CCA GGA ACT AAT GAA GA | Wild type Forward | A | |||
40303 | GGC TGA GCT CTG AAG CTA GG | Common | A | |||
40304 | CAG ATC CAA ATC CAA GAA AGA G | Mutant Forward | A | |||
40305 | Fluorophore-1 | TGG ATG AGA AAT TAC AGT AAG AAA TTT G | Quencher-1 | WT Probe | ||
40306 | Fluorophore-2 | ACA GTA TTG ACT CTT TTG TTA GTG GCT A | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
40302 | 0.40 uM |
40303 | 0.40 uM |
40304 | 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 |