Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr3:138179333-138179433 101bp ATCTTCCTCACGGCGCTTC TGTGTATTGGGGAGGTAAACA
Mut= 95 bp
Wt= 101 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GGAACAGGGCATCGGATCCCATTACAGATGGATGTGAGCCACCATGTGGTTGCTGGAATTTGAACTCAGGACCTCTGAAAGAGCAGTCAGTGCTCTTAACCACGGAGCCATCTCTCCAGCCCAAATGAACTTGTATAATGCTATTATACACAGTTTTACATCACAAGtttcctcacatgcacacataggtcagggctgagtttagatagactaggctatttcagcaactaattcataaagcattttgtgaataaaaaagcagtttgacttaatttaaatatattttcccaatttctttcagagccatgtagaaaatccagccagtgcttcaagacttccaaagacaatcccttggtgaaaaataaggtaaaaagctggctgcccacagccagctttgtagcttactctgacttctgacttctggcacttgatcttcctcacggcgcttctcagaagtctctttcctaccattttacagctctattaaagTACAGCTAAGGAGACACCAAATGTTTACCTCCCCAATACACAATTTGATGCACACTGACAAATACACATATACAACTCCCCCACCCCCGCCCCACCCCCAGGAAGCAGCACCCAAAACTGAAGTGTGCAGGTGTCTACCAACTCCATGGGTTTCCTCATGTGTGTCCCCAAAT
This mutation is a 322 bp deletion beginning at Chromosome 3 position 138,179,375 bp and ending after 138,179,696 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44684 | ATC TTC CTC ACG GCG CTT C | Wild type Forward | A | |||
44685 | TGT GTA TTG GGG AGG TAA ACA | Common | A | |||
44686 | TCC AGC CCA AAT GAA CTT GT | Mutant Forward | A | |||
44687 | Fluorophore-1 | AGT CTC TTT CCT ACC ATT TTA CAG CTC | Quencher-1 | WT Probe | ||
44688 | Fluorophore-2 | AGT TTT ACA TCA CAA GTA CAG CTA AGG A | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44684 | 0.40 uM |
44685 | 0.40 uM |
44686 | 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 |