Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr2:122112065-122112182 118bp TGTTGGTTGCATGTTGAGAC AACAAGCCGATGCACAGTTC
Mut = 139 bp
Wt = 118 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
TTTATTCTAAGTGTAGGTAAGATTTTGGTGACTTACAAGAAAACAAAATATCTGAGCTTTTAATGTTGGTTGCATGTTGAGACAACATTTGGGTATGGCTAACGTGTAATGCTAGATTGGGTTTAcccttctgtaacaggatggtaagcagcagctaggaagaactgtgcatcggcttgttaagccgctggcttctcagtcatctggttactgtgttggagaatgccctctttccagccctcctgctgtacctgcgaggtggtaaccattcagacaaccttatgatttaaagggtcctgcgtatttataaactgacacttctttaactctaactcagatatttttaacactatggatggcacgcaggtagcgcaagtggacctggctcttctcagagaagattctgagcagctggagcccgccagtgtctcttcctttacatcgctgagagtgtctcaggacttggatgttctggtgctggtcagctcagccagtactgcagttgctcttaacctacatttgtatttcaggtaggtagatggctcaaatttctcatttgaggtaaatagacccataaaaggtttaaaatatactccttagctttttttaaaagcctctttgctaTGCAGTTAGATCTCATTATGGCTCCTACAGTAAGACTGTTACCATGCAAGAGTGTGTATAAATGATTGTGGCTGCCTTTATATTTTGGCTTGTATATTAAA
This mutation is a 499 bp deletion beginning at Chromosome 2 position 122,111,622 bp and ending after 122,112,120 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
50154 | TGT TGG TTG CAT GTT GAG AC | Common | A | |||
50155 | AAC AAG CCG ATG CAC AGT TC | Wild type Reverse | A | |||
50156 | AGG CAG CCA CAA TCA TTT ATA C | Mutant Reverse | A | |||
50157 | Fluorophore-1 | TCT GTA ACA GGA TGG TAA GCA GCA | Quencher-1 | WT Probe | ||
50158 | Fluorophore-2 | TTT ATG CAG TTA GAT CTC ATT ATG GCT CC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
50154 | 0.40 uM |
50155 | 0.40 uM |
50156 | 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 |