Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr14:79386924-79387018 95bp CTGTCCCATCTGTTGCACTG TTCATGCCGAGAATGTGAGT
Mut= 95 bp
Wt= 95 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CAGTGGTAACTAAGTCACAGTCATATGGCTTTAAAAGTCTTTATTCTGTGTCAGTGTTGGTTGATGATTATGCATTTATTGAGTTTATTCTTCATTTATGTCTCTATCTCTTACAatagtggagatgctacatgtcaaggcatatataaccgcacttaatgctgaaattaatatagacagaagagcatgtcatcacattttaaactttggatcttgaattgataagatataataaaacttggttcattattttataaaataacctgctgtttccttttagaaatgttatgaacagaagcagtacaaaaatggcctcaagttttgcaagatgattctttctaacccgaagtttgctgaacatggaggtactgtcccatctgttgcactgtgctttgggggaacacaattctaaagccaggcaggacttctgaaagtgcaggaggactcacattctcggcatgaatgtttagcatttcttggttatgtttcgagtgaatcttaggttgagctagtttcatgctctagtatgtgcagtgccttgacttttaatgaggaagtcggtgagagacaattggatgaggtagtgtgcatgttaacatccctctcaGAAGTGCTTCAGATACCCGATGAGCTCAGACTTTGGAATAATTGTTTAGATTACCAGTTATACACACCAATTCAAAAAATCTTGAAACTCTAGTTTCAACTCTTAG
This mutation is a 492 bp deletion beginning at Chromosome 14 position 79,386,780 bp and ending after 79,387,271 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
39856 | CTG TCC CAT CTG TTG CAC TG | Wild type Forward | A | |||
39857 | TTC ATG CCG AGA ATG TGA GT | Wild type Reverse | A | |||
39858 | CTG TGT CAG TGT TGG TTG ATG A | Mutant Forward | A | |||
39859 | GCT CAT CGG GTA TCT GAA GC | Mutant Reverse | A | |||
39860 | Fluorophore-1 | CAG GAC TTC TGA AAG TGC AGG | Quencher-1 | WT Probe | ||
39861 | Fluorophore-2 | TTC ATT TAT GTC TCT ATC TCT TAC AGA AGT G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
39856 | 0.40 uM |
39857 | 0.40 uM |
39858 | 0.40 uM |
39859 | 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 |