Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr8:15069061+15069141 81bp ATGCCTGCTCCTGAAGGACT CCCAACATACATCCCTTGCT
Mut= 82 bp
Wt= 81 bp
Fam=Mut
Hex=Wt
Wt Sequence: (deleted region in lower case)
TCGTTGTCTGATACCACTCTCACCAGTCCTAAGTGTTAGCACAGGCACCTACCATTCCTTGAAGACATCAGTCCATTCTTGTATATATTGGTTCTTCACTTTGACAGGAAGGGATGGCTCTAATCCCCCAGTTGTAAGGCTGACACTTCTTCTCTGTGATCTCTCCACCTCTACATTCAGGGATCCTCTCCAGGGCCCCAAACATGCCTGCTCCTGAAGGACTCTAAGGAgtgcccaagtatttcagtctctcatgccaaagaaagcaagggatgtatgttgggcatagctctattaataaaccacagccgcctgctgttctttgtagctttctgtgtatactgacacatactgaaaagaggagcgcacaagtagaaaacgttgtttggcttttccccatcatcttttatcccttcccactgagggatgggtacacagagggggcctgtctagtctgtcagtgtcaccaaatctaccagttgtcctatgaccaacggagcttcttggctctcttaggtaccggtctcaagcggcttcttacggagaagctaagaggcagcgtttcctcagtgagctggcccagctggaggagaatgtccacctggcccggtcacaggctcgggacaagctagacaagtacttcatggagcagacggtaggaggacctgcaagctcctccctcggggtgtagaaaaggagagtttgtctgcacgcaaatggtctaatgcttcactaaccGTGGGCTGCTCCTGCCACACCCACTCTGACCACCCTCTCTTCTCCCCCATAGGTGGAAGACAATCTGGCGTGGGAGAGACACTCATTTGAAGACCGAATGAGCAGGGCCCCTGAGATCCTGGTGAGGTTAAGGTCCCACACCATCTGGGAGCGGATGTCTGTGAGGCTCTGTTTCACTGTGCAAGGATTCCCCACGCCTGTGGTGCAAT
This mutation is a 508 bp deletion beginning at Chromosome 8 negative strand position 15,069,595 bp and ending after 15,069,088 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
38450 | ATG CCT GCT CCT GAA GGA CT | Common | A | |||
38451 | CCC AAC ATA CAT CCC TTG CT | Wild type Reverse | A | |||
38452 | CAC CTA TGG GGG AGA AGA GA | Mutant Reverse | A | |||
38453 | Fluorophore-1 | TAT TTC AGT CTC TCA TGC CAA AGA | Quencher-1 | WT Probe | ||
38454 | Fluorophore-2 | TAA GGA GTG GGC TGC TCC T | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
38450 | 0.40 uM |
38451 | 0.40 uM |
38452 | 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 |