Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr9:51825862+51826026 165bp AAGACTACCCGAAGAGCATCC CACACGCACAGTCTTTCAGC
Mut= 166 bp
Wt= 165 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CTCCTCCAACATCAGTTGACATGGATATCCCATTCTTTGGCGAGTTCCATGCCTAATATCAATTTTTTTCTCTTTTATATAATCTGATTCGGCTGAGTTTTACCTGTATCCTTAAAAAAAGAAAAATGAGAAAAGAGAACATTTAAGTATATTGACAATGTTatcaagataggtttggtttttttttttttgttttgttttttttaatgagctattagtgaatctcatttgactcagtgttgttttggattatcacatttgattagatacatcgctctagaaaaggagaaagactacccgaagagcatccccctcaaaatctttgccaaggacattggaaactgtgcctatgtaagtgtttctaagccagtaaacgaaatccaaggtccctgtataagagcaaaaacattgtggatgaaaaaggagacaggcaggctgaaagactgtgcgtgtgtcctatagaAGGCAGGCATTTAGAGGTCCTGTGGTGGGGGTGTGCATCTATATGTATCTTTAAATGCATGAAAGTCAAGTTCTGGAGAGAATGGAAAAAGGGACATTCCTGCAGTATTTAACATGTAGCCCTGGATAGAACATTGCTGGTCAGAAATCTCTTCTCGGGGCAT
This mutation is a 301 bp deletion beginning at Chromosome 9 position 51,825,735 bp and ending after 51,826,035 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44447 | AAG ACT ACC CGA AGA GCA TCC | Wild type Forward | A | |||
44448 | CAC ACG CAC AGT CTT TCA GC | Wild type Reverse | A | |||
44449 | CGG CTG AGT TTT ACC TGT ATC C | Mutant Forward | A | |||
44450 | CCC TTT TTC CAT TCT CTC CA | Mutant Reverse | A | |||
44451 | Fluorophore-1 | CCA AGG ACA TTG GAA ACT GTG | Quencher-1 | WT Probe | ||
44452 | Fluorophore-2 | ATT GAC AAT GTT AGG CAG GCA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44447 | 0.40 uM |
44448 | 0.40 uM |
44449 | 0.40 uM |
44450 | 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 |