Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr16:94501187-94501291 105bp GCATTCAGCTTCTTGATGACTC GCTCACAGTTGACTCTGGATG
Mut= 100 bp
Wt= 105 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GGGAACACTTTAAAACGGCCTTTTTCTTTTTCTTCTTTTGTGACCTGGGGTTGAGGGGGTGGCTGTCCTTAAGCACCCTTCTAACCCTGAGGCTTTTTACATTTCATGTCTTTCGGCCTGAGAGAGCTTTGTCAGCCAGTGTAGGTTGGGAGCTGATTCGGAGTGAGCTGTGATgtccgtttgtccctaatatgtgtacagggtggttgatgagtcagtctggtggccactgaaagatgaaatggctattattccttcttgttttagaaagaagtttccatggtttatagaacaattactcaacttggttttcagttcttatttgtaccagtgcaactggaataagtgacagccccctttccttctgtttctgatgtctttgtctctgttctccatagagagcttcacttcccaaattcttcattagaggacatctgaactccactaactgcgctatcacgcagcccctgacaggagagctggtggtggaacactcggatgctgctatccggagcatagagcttcagttggtccgggtggagacctgtggtaagacttctagagtatctaggtatctacacttgtactgtcagccgctaaaacaccccttcctgtactcttaacatggcagggcacatcacatcataacagagctattctgatggcccctgtggcctactcaccccaaaattcccacttgcagataatcactgaaatactaattaggtccccagcaccttaaatcttggaagcatggtaaaaccagtgtggtgatgtttgagaacatgcaaagtagaagagaaggtaactcccagtcctgaagaagaaattggagaagtgggtgaatgtgttgatcctggttggcacagtattgggcagtttggtgaccagtttgctttggtgtcatggtgtcctgaccccatgatgtttcctctttctccagggtgtgcagaaggctatgcacgagatgccacagagattcagaatattcaaatcgctgatggggacatttgtagaaacctttctgttcccctgtacatggtcttccccagactgttcacctgtccaacactggagaccaccaacttcaaagtgggtaagcagtccaggcctggcctgtggagtctaggaagggtctgggccacagggaagaagcgccttgcacgcaggctgcagctttttttttttttttttggtttttcgagacagggtttctctgtgtagccctggctgttctggaattcactttgtagtccaggctggcctcgaactcagaaatccgcctgcctctgcctcctgagtgctgggattaaaggcgtgcgccaccacgcctggcttagcattcagcttcttgatgactcaaaaaagggtaagaagctgcgtaagtagGTGTGTGTAGGCAGTAGAGCCTGCTGCAAGGCCCCATCCAGAGTCAACTGTGAGCCAAGCAGAAATACCGTGAGTCTATAGGAAAAGCCTTGACAGTTGTCAGGTAAGTAGTATGTGAGTAAATATCCAACAGGAAACAACTTAA
This mutation is a 1215 bp deletion beginning at Chromosome 16 position 94,501,242 bp and ending after 94,502,456 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44111 | GCA TTC AGC TTC TTG ATG ACT C | Wild type Forward | A | |||
44112 | GCT CAC AGT TGA CTC TGG ATG | Common | A | |||
44113 | TGT CAG CCA GTG TAG GTT GG | Mutant Forward | A | |||
44114 | Fluorophore-1 | AAA GGG TAA GAA GCT GCG TAA GT | Quencher-1 | WT Probe | ||
44115 | Fluorophore-2 | CGG AGT GAG CTG TGA TGT GT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44111 | 0.40 uM |
44112 | 0.40 uM |
44113 | 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 |