Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr12:84459472+84459564 93bp CCAATGAGCCATCTCTCTACCT TGTATTTGTCACACAGCCTTCA
Mutant= 110 bp
Wild Type = 93 bp
AGAATCAAACCTGGGTCCTCTGGAAGAACGGTCTGTTCCTCTTGCCCAATGAGCCATCTCTCTACCTACAAACCTGGATTTTTGAGATtgcagtttgccaggatttttaaacagtctgaaggctgtgtgacaaatacattcttataagaaggatgttcctgaatgaatatattctattttattttctgacagcccatcacaagctctccaccgaaatggatggctgagatagagcgtgatgacattgatatgttgaaaggtatgcgatgctggtgtctactctcctctttttcgacttggTTTGGGTTGGCTTGGGTTTTTGAGACAAGGTTTCTCTGTGTAGCCCTGGCTGTCATGGATCAGGCAGGCCTGGAACTTGGAGTTGTGCCTGCCTCTGTCTCCTGTGT
This is a 212 bp deletion beginning at Chromosome 12 position 84,459,515 bp and ending after 84,459,726 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
47608 | CCA ATG AGC CAT CTC TCT ACC T | Common | A | |||
47609 | TGT ATT TGT CAC ACA GCC TTC A | Wild type Reverse | A | |||
47610 | CTG CCT GAT CCA TGA CAG C | Mutant Reverse | A | |||
47611 | Fluorophore-1 | TGC AGT TTG CCA GGA TTT TTA | Quencher-1 | WT Probe | ||
47612 | Fluorophore-2 | TTT GAG ATT TTG GGT TGG CTT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
47608 | 0.40 uM |
47609 | 0.40 uM |
47610 | 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 |