Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr5:135967847-135967972 126bp AGCGTTTGCGACGATGACT CAGGAAAATGGGCCCTCTG
Mut= 117 bp
Wt= 126 bp
Fam=Mut
Hex=Wt
MUT Sequence (insertions with carrots ^g^ (T insertion):
tcccgattccccactgcaccagcttgtctccatttttttttttttttttttttttacagAACTGAGGCTGGTAGGGGGCCCGAGCCGCTGCCGAGGGCGCCTCGAGGTCATGCATAGCGGCTCCTGGGGCAGCGTTTGCGACGATGACTGGGACGTTGTAGATGC^T^TCCATCTGGGCTGGGGACAGCACAACTGCGGCCACCACGAAGACGCCGGAGCGCTTTGTGCAGgtgaagctgtggcaagagaggaggtggagccggagtggtgggcggggcccgacccataggaggtgcgggcagggtttatatggaggaaa
This mutation is a 4952 bp deletion beginning at Chromosome 5 position 135,962,986 bp and ending after 135,967,937 bp (GRCm38/mm10). In addition, there is a single bp (T) insertion at the deletion site.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
47167 | AGC GTT TGC GAC GAT GAC T | Common | A | |||
47168 | CAG GAA AAT GGG CCC TCT G | Wild type Reverse | A | |||
47169 | CTC TTG CCA CAG CTT CAC CT | Mutant Reverse | A | |||
47170 | Fluorophore-1 | AAC GTG GTG TGT CGT CAG C | Quencher-1 | WT Probe | ||
47171 | Fluorophore-2 | ACA ACT GCG GCC ACC A | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
47167 | 0.40 uM |
47168 | 0.40 uM |
47169 | 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 |