Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr13:119702361+119702480 120bp CAGGTGGCTCTTCTGTTGC TGACTTTGGGATAGGCTAGGA
Mut= 118 bp
Wt= 120 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GCTAGTATGTTATGACTCATATTTGAAAATCAGTGATACAGTCATATATAGCTGAGTGTCTAAATCCACATTTCAAACTATTAAGAAATTTAATTAGACAGGATTTGGGGTCACTGTTTAAGGACACAAGCAGGTGGCTCTTCTGTTGCCACCCGCCctgctctctttgcagtgtagacataaacttgttttgtttcttaaaattaataagtaaataaagatacgagcatcctagcctatcccaaagtcaaggcacagtttctggcttctgtggatgccatcatttcttggtcttctctttcagagggaaaggataaagattttaccctgaatgattttggcttcatgatctttcactcaccatattgtaaactggtgcagaaatctctagctcggatgttcctgaatgactttcttaatgatcaaaacagagataaaaacagtatttacagtggactggaagcctttgggtaagaacgctgttatgatgtgcttatttctattttataaatatttacatatatatagacctagagatacagtgagaaaacattctattctagttgagtggttgtggtccacgcctttacttccagcactcgggaggcagaggcaggcagatctctgaatttgaggacagccagggttacacagagaaactccgtctcaaaaaaaccaaaaaactttatttaatgaaaaccagttCTAGAGAGGTAGTAAGTAAATGGCTTATCTACACAAAATCATTAGTCATTTCTACTTAAGATTCTGGTGTCTGTTTTTAGCTTCAGCCTCATTGTTAGCGCACAATACTGTAATCACAGCCGCACCAGTGTACACCAAGCTG
This mutation is a 548 bp deletion beginning at Chromosome 13 position 119,702,388 bp and ending after 119,702,935 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
45646 | CAG GTG GCT CTT CTG TTG C | Common | A | |||
45647 | TGA CTT TGG GAT AGG CTA GGA | Wild type Reverse | A | |||
45648 | TGA GGC TGA AGC TAA AAA CAG | Mutant Reverse | A | |||
45649 | Fluorophore-1 | TGC TCT CTT TGC AGT GTA GAC ATA A | Quencher-1 | WT Probe | ||
45650 | Fluorophore-2 | CCC TAG AGA GGT AGT AAG TAA ATG GCT T | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
45646 | 0.40 uM |
45647 | 0.40 uM |
45648 | 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 |