Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr7:100831468+100831600 133bp CAGGTAGGTACACCTGGAGGA GCAGGAATTGCTTCAGTGACA
Mut= 126 bp
Wt= 133 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CTGGGTAAGGGGCCAGAATGAGAACAGAGCAATAAGAAATGAGGGAGTAGGGGGCATCTCTACATGTACTTTCCACACAGGCAACTCTGAACCCAGGCAGGCTGTCAGCTGAAAAGGCCCCAGAGAGACTGCTTCTAGCCTCTTTTTTTGAATAGAACCCTGGAACCCAGAGAACTAACACATATCCAAATCATACTTGCAgcccatgggcaccaggactgagggagctgcctggggtcaggctctgagggtagaaccttgatctcttgtctgtcctatctgtgtttccagctggattcaacctgttcttgtttgaggaactgttactgggccctgctgtcttcaagcaatgctgttctaccaactaactagacgtctcttttattgccttggtacaggatataccgcagggacaccatacaaggtcccaccgacccagagtaatactgctccacccccctactccccatcacccaatccctatcagacggccatgtatccaatcagaagtgcctatccccagcagaatctgtatgcccaggtaggtacacctggaggacctcttctccgggagcctccagccaggatggggtggcatgggatgaggaagaaaaaacagagttaatgggcctgGCCAGCTATCACTGTAGTGTCACTGAAGCAATTCCTGCAGGGCTGCCTGGGCTGCCTTACTCCCTAAGGGTGAAATGTCAAGTCCTTTAGGTTAACAGAGCCCCAAATGTCAGAGGGCCATTAGAAATCACCCAGCATAACTCTCTTGTTGGACAAGTGGGGAAGCTGAGACTGCAGTGGAGAAAGACTTGTCTAAG
This mutation is a 432 bp deletion beginning at Chromosome 7 position 100,831,131 bp and ending after 100,831,562 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
47364 | CAG GTA GGT ACA CCT GGA GGA | Wild type Forward | A | |||
47366 | GCA GGA ATT GCT TCA GTG ACA | Common | A | |||
47367 | AAG GCC CCA GAG AGA CTG CT | Mutant Forward | A | |||
47368 | Fluorophore-1 | CTT CTC CGG GAG CCT CC | Quencher-1 | WT Probe | ||
47369 | Fluorophore-2 | CAA ATC ATA CTT GCA GCC AGC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
47364 | 0.40 uM |
47366 | 0.40 uM |
47367 | 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 |