Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr11:50140636+50140749 114bp GGGGAAAAGGATTGTAGATTTACTG CCTCCTGCATGTTTCAGCTC
Mut = 103 bp
Wt = 114 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
CTGTGGGAAGAGCACTGTGCTTCCGTTTCCACTCTCCGTGCACTGCTCAGACAGATGCTGGGGAGTGGGGGAGGCCAGCTGAAGACAGTAGCTGTTTGCCTGCCCTGCAgggactctgtcaacaacgggacaaagaaatctgataaatttacctgtttacagccgccttgtagattgtctctagaagaacaggagtactcttggtaggggagggaggactctcactgtaatagaaagtgtaaacaggagaggctcatccttgccttgggaggagggggtcacttgaatagagagagagcactggggacccagccctctgcttgagtctgagctctgatttcatccactctcactgaactaggttcttcccgaaaagagatcaccaaacactgggaatggctggaaaacaacttgcttcagacactgtccatcttcgacaacgaggaggacatcactaccttcgtcaagggcaagatacacgtaaggcccatgtcccaccgacaaccaacccagctccctttgtgaagtgctagcggccacacacagttgttgctccatatctgtgggatacaatgaattccatggattcaaccaactgtgtgtcgaaaatatttggggaaaaggattgtagatttactgaacactgtcagtctttcctaggcaatacagaagagcagagcatccctgcTGTGGTGCGTGTTGTAAGATGAGCTGAAACATGCAGGAGGGGGGACAGGCAAACTATCAACATACATAGATTTATATAT
This mutation is a 579 bp deletion beginning at Chromosome 11 position 50,140,131 bp and ending after 50,140,709 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
51362 | CCT CCT GCA TGT TTC AGC TC | Common | A | |||
51363 | GGG GAA AAG GAT TGT AGA TTT ACT G | Wild type Forward | A | |||
51364 | TCA GAC AGA TGC TGG GGA GT | Mutant Forward | A | |||
51365 | Fluorophore-1 | AAT ACA GAA GAG CAG AGC ATC CC | Quencher-1 | WT Probe | ||
51366 | Fluorophore-2 | AGG CCA GCT GAA GAC AGT AGC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
51362 | 0.40 uM |
51363 | 0.40 uM |
51364 | 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 |