Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr12:73292209+73292347 139bp CTGCTTGCCATGTGCATC CCTGTTTGTGCTTGCTAAATACG
Mut= 124 bp
Wt= 139 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CACATAGAGCCTAGGAATCGTCAGCTCGACAGTACAGGCAAAGCCATGGTTACAGGGCCCACATCACTGGAAGGAGAGGGAGTTGGGGAAATGGGGCAAGAGTAAGTCATTGAGACTTAGAACAGGAAAATTAGTCACTAAAGGGAAATATTTAAATCACTGAGTGCTGAAATGCAGTCAGCAGACagtgcaaagtgagggacttgtgttttaagtaaagtttctttctttctgacttcacttttcattttaaaagcatttcatttgacttaagtacaagtcggtaaatacagaacagtgatttctttgatgtctgcagctctgcttctcagagacggtccctacaagcatctgactgaagaaatgacggatcatgaaaaagcaagcttggaggaaacctaacggtgttgttttatgttttagtttcctgctgctgtttgttgctctcctggcttcttactcagtccatctcctgcttgccatgtgcatccacaccggtgagtaacaccatgctgctcacttgacccagcctcgtgcataagaaatgaatgtctcccctctgaTGTGTGAGAGTCAAACGAAAGCGTACGTATTTAGCAAGCACAAACAGGCCCCAGGCTTGGAACTCCAGCACCAACACGCCCCAAGCGTCTGGTTTCCAATGAGCATATATCATTAATGGTGTGGTGATTGGTTTTGTTTTCTTGTGTTAAGGACAAAAG
This mutation is a 377 bp deletion beginning at Chromosome 12 position 73,291,923 bp and ending after 73,292,299 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46131 | CTG CTT GCC ATG TGC ATC | Wild type Forward | A | |||
46132 | CCT GTT TGT GCT TGC TAA ATA CG | Common | A | |||
46133 | TGA GAC TTA GAA CAG GAA AAT TAG TCA | Mutant Forward | A | |||
46134 | Fluorophore-1 | ACA CCA TGC TGC TCA CTT GAC | Quencher-1 | WT Probe | ||
46135 | Fluorophore-2 | ATG CAG TCA GCA GAC TGT GTG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
46131 | 0.40 uM |
46132 | 0.40 uM |
46133 | 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 |