Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr7:19474494+19474610 117bp TTGCACTGGCACAATCATGT GCTACATGAAACCCAATCCCTA
Mut= 123 bp
Wt= 117 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TGCACGTATATGTGGAGGCCAGAGGAAACCTTGCCCGAATCAGTTCTACCATGTGGGCCCTGGGAATGGGGAAGAAACTCGTTAAACTTGGATGGTCGTTAAACTTGACAGCATGCACGTTTGTCCACTCCATCTCATTTGCCTGTTTGTTTGTTGTGACTTGTCCTCAACTCTGTAGCCCACTctcatgagtgcaattgctgaggtcaaaggcaagcgtcaccctcactctcatgttggggggaactcattttgggtggcaagccctgactctctcctcatcccccgcttctcactttgcgggtactgggtatgtgccccgcttcctgacaccttttctctccccaccctgcacagtgctggagatcctgggcctgatccagaggcgcgagctggcgcgagcggacgagcacatcctggagctggaggcggaggcgccgacggcggaggccgaggcgggcgcggggcggcgcgcgcgggacgtggcgctgctgtacgaggcgctgcagcgcgagctctgggcgctggtgcgcgagtcgctggcttccccgggacccggcaccggcgcggtgtcgcagctgggcctcgtgctgctgcaggaggaggcggcggacgcgcgccgcggccccggggcggcccgcaagctgcgcacgcactgggccgaggcggtggcgcgcgcagcgcgcgaacgtctggaggaggcggcggcgccgggcgcgcgcggggcgctggctgggcagctggaggcgctgcgcgcgcgcctgttggaggacgtggccgctgtgcgcggacgcctggcgcccgcctaccctgcggggctgcgcgccggcgccgtctacctgcgtggctaccacgaggcactggccgagtggctcggggcggccgcgcgccgcaggctcccgctggcggatcgttatgcgctgttgcactggcacaatcatgtgtaccccaggtgagcccgggtcgccccggGACGCGCTCACCTTAACCCCAGGCACCTTGTTCAAGATACTGGATTTAGGGATTGGGTTTCATGTAGCCCAGGTTGACTTGAGGTTTTCTGAGACAGGGTCTTGCTCTGTAGACTAGGCTTGCCCTAAACTCAGAGATCCGCTCGCTTTTACCTCTTGAGAGCTCCTATAAAAGCGAGCACCGCTGCACCC
This mutation is a 788 bp deletion beginning at Chromosome 7 position 19,473,755 bp and ending after 19,474,542 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
45591 | TTG CAC TGG CAC AAT CAT GT | Wild type Forward | A | |||
45592 | GCT ACA TGA AAC CCA ATC CCT A | Common | A | |||
45593 | CCA TCT CAT TTG CCT GTT TG | Mutant Forward | A | |||
45594 | Fluorophore-1 | CAG GTG AGC CCG GGT C | Quencher-1 | WT Probe | ||
45595 | Fluorophore-2 | TAG CCC ACT GAC GCG C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTP KAPA | 0.26 mM |
45591 | 0.50 uM |
45592 | 0.50 uM |
45593 | 0.50 uM |
Glycerol | 6.50 % |
Dye | 1.00 X |
Kapa 2G HS taq polym | 0.03 U/ul |
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 |