Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr4:43968866+43968968 103bp GGGAGCCTGAAGAGAGATGA CCGGCTGAAGTAATACAGATGC
Mut = 95 bp
Wt = 103 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
tgttgttcgttcctctgtcattccagcttccaaacagtttaataacgaggtcctgaaggCCCACAACGAGTACCGGGCCCAGCACGGTGTCCCGCCCCTGAAGCTTTGTAAGAAGCTCAACCGGGAAGCCCAGCAGTGAGTTCCCAGCAGCCATTGGCTCGGGCCCTTTGTGGAGCTGCTTCTGcagtcatgagggcccctgggtggttgaagggatgtgatggggtctcagatatagccagacttaaggacctactcttttgctggcccaggcaggcccctttgctcaatcttctaaaaatgggatggttgggaggcccaggctatacaagacctgtgtttctgttatccctctggtgaacccagatagggaaggaggctcagcagtacaaatctcagacgttggatgttggatggtgtgtgtgtcagggatggaggtgggggtgtccccgggcggggcctgctctgaggagatacattatgcaggtattcggaggccctggccagcacgagaattctcaagcacagcccagagtccagtcgtggccagtgtggagagaaccttgcgtgggcgtcctatgatcagacaggtaggacactcagctcctccccgctctgctctgctagatctaagatgaggcctctgggcgctcctcctggcactgcccttcacagttcaatgatggtagagccacaggcccaggaagggaaagaaaacccctgaattctctgcatctgttcctgggctgacctcagtagctgccccacagccagtaggctgggagcctgaagagagatgagtcagaggagacagctcgggtgcagaagtacaagtcagaggcgcgggCGCTGGAGACTGCAGCATCTGTATTACTTCAGCCGGCTGCGGGCACCGGAGCGTCCCGTGCTTTGCTAAATTATTTATTT
This mutation is a 659 bp deletion beginning at Chromosome 4 position 43,968,272 bp and ending after 43,968,930 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
49988 | AAG CCC AGC AGT GAG TTC C | Mutant Forward | A | |||
49990 | CCG GCT GAA GTA ATA CAG ATG C | Common | A | |||
50770 | Fluorophore-1 | AGC AGC CAT TGG CTC GG | Quencher-1 | MUT Probe | ||
50888 | GGG AGC CTG AAG AGA GAT GA | Wild type Forward | A | |||
50889 | Fluorophore-2 | AGG AGA CAG CTC GGG TGC A | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
49988 | 0.40 uM |
49990 | 0.40 uM |
50888 | 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 |