Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr15:89189870-89189960 91bp ACCTTCCAGGGGTCTTGGAG TTCTGAAAGTGGGCTGATCC
Mut = 108 bp
Wt = 120 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
AAGCTGCAGTAAGGTAGCCAGGTTCTCTAAAGCCCATGGATAGCCATATATCTGCGCAATCTGGCCTCCTTGACGCCATGACAGCAGCTGACCCGTGAGCACTGTGTAGTGGATTCTCCTGTCCCAGGCAGACTGAGTCAGGCTCcttcagggaaactgaaagtgattctctgtgttgattcatccaaggcccatgtgttgtctgcaggatcaggctaggagtctgctgggaaatagatcctcaagtctgtttgcccttgatcggatatttgtgccaacaaaggccagcccggtgcacccttgtaccacagccctcagccctgacaatgggaggagattctcccactgtgacttgtatggtgggactgcctctttccagggtgcctgggagacactcagttcagtttccgtatgcgtcagtgtgggggacaaaggagcctctggcaggtggatgatcggtcgtataacaacaaggcccccttggcattgcaggtaagtagtggggtgtctctctggtctgtgtcttctctcccttggtggggggctcctggctgtgaacctgagctttgtgtgatggaagtgctggccaggctcatcctgagaggtcaatgacagttgcaggtcctgctgattctgtcttggggttctccacctaggccagctcttatggggttcaccaggatcactgaagttgattctgtgcacccgatttcccttgctgggcatctgtgtggctaaataagctgtggttcaatgagcaaaacccggagggaccttccaggggtcttggagcactctcagattggctaaattcttTCTGAATCAGCAGATGTCCTCTAGACCGGATCAGCCCACTTTCAGAACTAGGGAATCCTAAGTCTCACTGTGCTAGGCAGAGGGCAGGCCCTAGTCTCAGGGGCAAGTGCCTCCTAGGGTCTCCTAGATTCTTAGACCCCACACATATACCCTTTCCTTT
This mutation is a 571 bp deletion beginning at Chromosome 15 position 89,189,967 bp and ending after 89,190,537 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
49651 | TTC TGA AAG TGG GCT GAT CC | Common | A | |||
49652 | CTG TGG TTC AAT GAG CAA AAC | Wild type Forward | A | |||
49653 | GAC CCG TGA GCA CTG TGT AG | Mutant Forward | A | |||
49654 | Fluorophore-1 | AGG GAC CTT CCA GGG GTC TTG | Quencher-1 | WT Probe | ||
49655 | Fluorophore-2 | AGG CAG ACT GAG TCA GGC TCT CT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
49651 | 0.40 uM |
49652 | 0.40 uM |
49653 | 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 |