Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr5:31532176+31532297 122bp GTTAACATATGGTTGCAGAGAATAGCC CCGCTTTCATTCATTTCACC
Mut = 137 bp
Wt = 122 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
ACATACACACACACTCACAATTGATAAATAAAAGTATGTATGGAGAGATGATTGGTTTGGATGTTTTGTTCTGATTGTGCTGAGATCAAATCCAGGACCTTGCTCATGCTAGGCAAGCATTCTGTCACTAAGCAACAACCTGTGACCCCCATCTCCTGGAGACCAGGTAGGCCTTGTAGGTATGAGCTTCTTGCCTCAGTTTCTGAATGCCTAGTATTATAAGCTGTATGTATAAGTATACACAGCCAAGATTGATTTCCAGGAGGTGATccctggcagtgctcgctgtgatgttagacactgagatctttgatctgatacagtccttactggatcaagtagtaattttagacctgactgagactgttcttttcctgcaggagaagagttagaatatgaatttgatgagcaaggacacagcacttggctctgcagggtgaggtatgttcttctcgtttgtattctagctgtgatttgagaccgggtcttgttttgtggcagtcttccctcttttgcctactgagtactgggttcatgtgctctttccttgctaatgtgtatgtttgaaagtagccagtttcaagttaacatatggttgcagagaatagcccaagagtaacttcactctgcactttaggctagttccaagtgtgggccttggTAGCTTAATAGCACTGGGAGCTGGGGTGAAATGAATGAAAGCGGGTGTAAATGACTTGAAGTCCAACTGAGGCAAGAAAGAGTGAAGTTACCTGAAAGCATGAAATGCAGTCATCATGGACACTCTTGATAGCTGTTTTCATGAGAGTCAGAGCATCCTGCTGAGCTCCACCCCCTGTAAACCAATGCAGGTTACCTGTAGATGATTCAACTGGGAAACAGCTGGTGGCTGAGGCCATTCACTCAGGAAAGAAAAA
This mutation is a 391 bp deletion beginning at Chromosome 5 position 31,531,863 bp and ending after 31,532,253 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48916 | CCG CTT TCA TTC ATT TCA CC | Common | A | |||
48917 | GTT AAC ATA TGG TTG CAG AGA ATA GCC | Wild type Forward | A | |||
48918 | GGT ATG AGC TTC TTG CCT CA | Mutant Forward | A | |||
48919 | Fluorophore-1 | CTC TGC ACT TTA GGC TAG TTC CA | Quencher-1 | WT Probe | ||
48920 | Fluorophore-2 | CAG GAG GTG ATT TAG CTT AAT AGC AC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48916 | 0.40 uM |
48917 | 0.40 uM |
48918 | 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 |