Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr12:81510760-81510896 137bp GCCGTATCAAAGAGGATGG AGGCCAAGATGGAATCAGA
Mut= 124 bp
Wt= 137 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
AATATCACCAACAGGATCAAAATCAAGTGTGGGCAGAAGCACTTATATCAAGACTCAACCATACTATTTCCCATGTTATATATATGTACATATATATTTATATTTCATCTAGCCAGTCATTTTGATCACAGTTGTGATCACAGTACCTGTAgtactgcctggttcgaattagcaatctaggtacttttgaactttgttcataatgacttattgactttgcccatgactgtgttttagcttacctcacagtctgaattattagtctctcttccccactagaccatttgcctctcctttcttctcctctaatctttctttcttcttcgtccctctctcctcactcttccatgattttctcagggctgggcttcaacatcgtcggtgggacagatcaacagtatgtctccaacgacagtggcatctacgtcagccgtatcaaagaggatggggctgcggcccaggatgggcggctccaggagggtgataagatcctctcggtgagaaatggcttcggctttctttgctatactttgctccctagaCCACATCTGATTCCATCTTGGCCTGCCTGTTTGGAAATGAAAATATTTAGAGTGTATCATTGAATAGGAAGAACATTGATCAGTCTTCCAACTTTACCGCTGGGATGGGAGGATAGTATTATTCTTGTTTGACAGACAAGGCAAATTTA
This mutation is a 391 bp deletion beginning at Chromosome 12 position 81,510,784 bp and ending after 81,511,174 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
40033 | GCC GTA TCA AAG AGG ATG G | Wild type Forward | A | |||
40034 | AGG CCA AGA TGG AAT CAG A | Common | A | |||
40035 | GAC TCA ACC ATA CTA TTT CCC ATG T | Mutant Forward | A | |||
40036 | Fluorophore-1 | TCG GCT TTC TTT GCT ATA CTT TG | Quencher-1 | WT Probe | ||
40037 | Fluorophore-2 | TGT GAT CAC AGT ACC TGT ACC ACA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
40033 | 0.40 uM |
40034 | 0.40 uM |
40035 | 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 |