Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr3:72960945-72961044 100bp TACTGCTGCGTCAGTCACGA CAAAGAACATCTTAAAATAGAGCCAAC
Mut= 99 bp
Wt= 100 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TGGTCAAGCAGTTATCTGGAGTTGACAATCTCTTATTTTTTTAATAAGCCCTAGTCATTGCTACAAAAGGTATATGTGACTGAATTAAATCTAGATTTTGAGTTACGTAACCTCACctgaacttgatagctatcatagcataagtattcaaaatcttcttattatcatgacgaagaggaaaaaaagacaaacgtaattattgagagtgcaaagggtcttaaaaggtgtgtgcttagagtattaatgataagaaaacagactaagtacatctgattgcattgccaattacgtttttgtttgtttgcttgtttgtttgtttaaaaatattttttccaggcaaaatgtgaagagcgtggctgttgctggcgtccatggaacaacactattattccttggtgcttctttgcagataaccatggctatactgctgcgtcagtcacgaatgacaacagtggtaagggtgctgccaccatgatagcccaATACATGTTCTAAGTTGGCTCTATTTTAAGATGTTCTTTGGAAAGAGCATCTTCAATAAGTACACACATCAAAACTTTTTTAAAATCTTTTTTTTATTAGATATTTTCTTTATTTACATTTCAA
This mutation is a 366 bp deletion beginning at Chromosome 3 position 72,960,985 bp and ending after 72,961,350 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
39759 | TAC TGC TGC GTC AGT CAC GA | Wild type Forward | A | |||
39760 | CAA AGA ACA TCT TAA AAT AGA GCC AAC | Common | A | |||
39761 | TTG CTA CAA AAG GTA TAT GTG ACT GA | Mutant Forward | A | |||
39762 | Fluorophore-1 | CTG CCA CCA TGA TAG CCC | Quencher-1 | WT Probe | ||
39763 | Fluorophore-2 | TGA GTT ACG TAA CCT CAC ATA CAT GTT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
39759 | 0.40 uM |
39760 | 0.40 uM |
39761 | 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 |