Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mut = 117 bp
Wt = 122 bp
Fam = Mut
Hex = Wt
>chr3:69223001+69223122 122bp CACTGGAGAAGGGCTGGAT CCATTTCCACTTCATCATTGTC
WT Sequence (deletions in lower case):
GAAACGATCTTTAAGGAAGAAAAAGCCTAAAATGGGTCTGCTGAATTCTAAAAACCCCCAAAGCAAGCAAGCCcacattcttctcctcggacttgactcagctgggaaatctactctgctttaccggttaaagtttgctgagactctctcaaccatcccaaccatcggcttcaacgtggaaatggtccagctgcagagcagtctcacactcaccgtgtgggatgttggaggacaggagaagatgcgaacagtctgggactgctactgtgagaacgctcaagggctgatgtacgtggtggactgttccgaaggcaaaaagcgactggaagactctcgcaaagagttcaaacacattttgaagaacgagcacatcaaaaacacaccggtcgtcatactggccaacaaacaggacttgccgggagctctgagcgccgaagacatcaccaggatgttcaaggtgaagaagctgtgcagcaaccggaactggtatgtgcaaccctgctgtgcggtcactggagaagggctggatgacgggttcaggaaattaaccgagtttctgaaaagctaccgaaggacaagagagaccttagcaatcttCAAGCAGAAATGAGACAATGATGAAGTGGAAATGGGAAA
This mutation is a 524 bp deletion beginning at Chromosome 3 position 69,129,897 bp and ending after 69,130,420 bp (GRCm39/mm39).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
58118 | CCA TTT CCA CTT CAT CAT TGT C | Common | A | |||
58119 | CAC TGG AGA AGG GCT GGA T | Wild type Forward | A | |||
58120 | GGG CCA AAA GAA ACG ATC T | Mutant Forward | A | |||
58121 | Fluorophore-1 | ACG GGT TCA GGA AAT TAA CCG | Quencher-1 | WT Probe | ||
58122 | Fluorophore-2 | CCA AAG CAA GCA AGC CCA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
58118 | 0.40 uM |
58119 | 0.40 uM |
58120 | 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 |