Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr11:59186041-59186161 121bp TGGGGACAGTGGTATCTGTTC GGAGAAGAAGAGAGCTTTCCAG
Mut= 106 bp
Wt= 121 bp
Fam=Mut
Hex=Wt
The WT Reverse primer (primer 65355) anneals over the nucleotide sequence containing mouse genomic variations rs214496484.
Wt Sequence (deletions in lower case):
CCAGCAGGAGAATCTCTCTTGCTGTTGGGTTCACAGCTTGATTAGAGTCTGCATCATAGTGAAGGCTGAACCTTCTTCCTGCCTGGTGTATGGAACCTGGCCATTTCCAATCCTTCAGCAAAGACAATGGGGACAGTGGTATCTGTTCCAGCCTAGATCTGGCCATAGCAGTCCTGTgtagggtggagaggggccaggccaggcagggatgggttttgggtaccttctggaaagctctcttcttctccagattactactttgtgaccagggagatgatgcagcgtgatattgcagcaggggacttcattgagcatgctgagttctcagggaacctgtacgggacaaggtgggctgagtttggtgtgggcccagcttcatggatcagggtaattccagaactgttaacaaaagaaagcactctagcatgtcacctctgacctcacagtgccctcccagtctcccgcctagggctgggatggcttgctaaccccctagtctctgagcatagctgataggctccagggaacaagcaggcccagtcatgccccaggctcatctgtcctcaagccatctggcgaggcaaagagccacatgaggcacctcacGTGGAGAGGTAGGCAGTGCCAGGCAGCCCAGGCCCAAACCTTTTTGGCTTTCGGCTACAGGACAGGTACAAAGGGCTGTGATGTCAGAGGGCCTGACAGGGTTGACTATATGCTTGGAAGTTCTGGAAAGTACAGAAGGTGCTGGGTTCACGAGAGCCTCTGCACTACCCAGGCCAGGCCCAGAATGGGCCTTGGGGTGGAAAGGGC
This mutation is a a 429 bp deletion beginning at Chromosome 11 position 59,185,683 bp and ending after 59,186,111 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
64355 | TGG GGA CAG TGG TAT CTG TTC | Common | A | |||
64358 | Fluorophore-1 | AGC AGT CCT GTG TGG AGA GGT AGG | Quencher-1 | MUT Probe | ||
65355 | GGA GAA GAA GAG AGC TTT CCA G | Wild type Reverse | A | |||
65356 | AGC CGA AAG CCA AAA AGG T | Mutant Reverse | A | |||
65357 | Fluorophore-2 | CAG GGA TGG GTT TTG GGT A | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
64355 | 0.40 uM |
65355 | 0.40 uM |
65356 | 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 |