Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr11:99591312-99591424 113bp GCAGCCAGCCAGGGATTAT CAGGCCATGGTGTCAGAAGT
Mut= 118 bp
Wt= 113 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletions in lower case):
GAAGATTATAGTGAATATGGGTGATCCAGGCTCATGTTTCCAATGAACGCTAATGACTACTTCACTGTAGTCAAGAAGTCTAATTGAACACCAAATAAAATCAGCTTTTTACGGGCTTTCAGAAGTCTGAACAAGTAACTCAGAAACAAACAATTATCATTTAGAGAAAAACTACATAAACATAAACAAGGAAGACACTTTGGAAATTAGGGAAACAGCAGCCAGCCAGGGATTATAaaaagactggagtggcactcaccacacaaattcaaaaaacttctctaagcaaacaaaccttcagcacagatcaacttctgacaccatggcctgctgcgctactagcttctgtggctttcccacatgttccactggtggcacctgtggctccagctgctgccagcccagctgctgccagcccagctgctgccagcctagctgctcccagtccagctgctgccagcccagctgctgccagtccagctgttgccagcccagctgctgccaatccagttgctgccaaccttgctgctcccagaccagctgttgccagcccagctgctgccagcccagctgctgtggaactggcagtggccaggagggtggcagtggagccgtgagctgccgtgttagatggtgccgccctgactgccgcgtggagggcacctgcctgcccccctgctgtgtggtaagctgcacacccccaacctgctgccagctgcaccatgcccaggcctcctgctgccgtccatcctattgtggacagtcctgctgccgcccagcctgctgctgctactgctgcccacccagctgctctgagtctaactgctgtgagcctacctgttaagacccaggttgctgattcccaaaggatcttgaattttgatatggcttgtgtttggggcacaaatacttggccttactaaacatttttttccatctaatactggaaaggctttgatatcctctatgaaaataataaaatacaaacttgatccccaaacaagaaaatcaatttaccttgggattgtccctattttccaaggcttggtgctgtcaaggccatggaagattcttctacacgaagatagttttcattcttcttccattgttctgtaaattcctcttataaatgaaggctaatgtatccaaaataataaattatatctgttggcatatcaaaacttgcttctttttatttagtgtgcaaataatagatacttcatggagatccatctaaaagatttctgagtttgattctatacacttatctattttgtgtggggagtggggcatgtaagtagagatcagtctctccttccaccttatgattctgaggactgaactccagtcatcgggcttagaggcaagtgcctttacccactgagctgccttgtcagccctgtttttttatattcttcttatacctgttaggaaatgggatgaattccaacagtggttagaccccaaaaaagttgtagcattgccctggtcttgttttattttgctgccgtcaataaacattgtgtccaaaaacatggggagcaaagaggttgtttggcTTATACTTCCAGAACACAGTTCATCACTGAGGAAAGTCAAGGCAGAAACTCAGAGTCAGGAACTTAGTGGCTTGTTTTCTAGCTCACTTGTGGGATCAATTTTAGCTAGCTCTCTCATAAGGCCAGCCCCTCCTCAATCAATTAATAATCAACATAGTCTCTTGTAGGGATAGAGACAGTCACTTGGTTGAGGGTCCCTTTCGGGCTATCACAGGCTGTGTCT
This mutation is a 1322 bp deletion beginning at Chromosome 11 position 99,590,083 bp and ending after 99,591,404 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
64388 | GCA GCC AGC CAG GGA TTA T | Common | A | |||
64389 | CAG GCC ATG GTG TCA GAA GT | Wild type Reverse | A | |||
64390 | TGA TCC CAC AAG TGA GCT AGA A | Mutant Reverse | A | |||
64391 | Fluorophore-1 | CAA ACA AAC CTT CAG CAC AGA T | Quencher-1 | WT Probe | ||
64392 | Fluorophore-2 | AAC TCA GAG TCA GGA ACT TAG TGG 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 |
64388 | 0.50 uM |
64389 | 0.50 uM |
64390 | 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 |