Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr1:87185123+87185226 104bp CCCAGGGTAGGAGAACATCA GCCTGTAAGTGTGGCTGTTG
Mut= 120 bp
Wt= 104 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
ATCCAGCACAGACTACATGAATGCCAAGGTTGTACCAGAAGTGCTACCCCACCCAGCAGACTCACATTGGTACAGGGGACTCCTCCTTTTGTCCATGTCCAGCCCTTATTTTTAGGTTCCATCgtccttggggtcagattctttttgaaggtccaagtggaaggggtatgccaaacttgagacacagcagggaggaggggagagtgtgggctcaggtggtcacagttcatggctgctcaaatagttccatgtgctagaaggcgggccaagtggagagtggttgaaagatgaggcccagcccccccccccaataaagcgaaagatccgcaagagggcgcagcgaggctcaaaggattgtttctcagtaggacctggacgccctagacctcaagcccccctcctccaggacccaccggagccaggtgaaactaaaagactccaagggctgaaagcgaggtgggcctcaacgtgaagataactaaaacttttgcttcatgacgccgtttctgccattctagtacaatgtggagaaagacatcaaggcgttgccaacacgacccgggctcacggccgcatagtcggtggcagcacggctccctcgggggcctggccttggctagtgaggctgcaacttggagggttgcctctgtgcgggggtgtcctggtagcagcatcctgggtgctcacggctgcacattgcttcgccgggtacgtcgtcgagattccaggcacctgctcagcagggccacccaatccgatgggagtctaagcacccatgggccaaagcagagcagagcaggtctctgctgccccctagcggaagctagctcccaacctcgggccttaaaatgccggttgcgggtgggggtgggggtgggggtgagggatcccagggtaggagaacatcacactgaattttcccactgcagggaatctgcaccttcccggtctccctctctttcccttggGTCACAACAGCCACACTTACAGGCTCACTGGATGGTGTGGGTGCATGAGGGTCTGGGCCTCTGAGGGGGCCTCACGCCACCCTCTGTCCCTGATTGGTGTCACCCA
This mutation is a 845 bp deletion beginning at Chromosome 1 position 87,184,358 bp and ending after 87,185,202 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
40354 | CCC AGG GTA GGA GAA CAT CA | Wild type Forward | A | |||
40355 | GCC TGT AAG TGT GGC TGT TG | Common | A | |||
40356 | GGT TGT ACC AGA AGT GCT ACC C | Mutant Forward | A | |||
40357 | Fluorophore-1 | TTC CCA CTG CAG GGA ATC T | Quencher-1 | WT Probe | ||
40358 | Fluorophore-2 | CCT TAT TTT TAG GTT CCA TCG TCA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
40354 | 0.40 uM |
40355 | 0.40 uM |
40356 | 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 |