Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr12:80947685+80947809 125bp GCTGAGAACCCAGAAACTTGA CAGGTGGAGCATTTCTGAAG
Mut= 126 bp
Wt= 125 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GTGAATTGTTTTCCCAGAAAGATGTTAGAAGTGGCCAAGAAGCAGTGGCTTCTGTTAAGAAGTTAAAGTAACTGCTGGCTGGAGTTGATTCTCAGTCATCCCTAGCTTAGGGTTATAGTATCTGAGTGTAAGGTATATCTTAACACTTCCATGATTTTTTTTTTAAATttattttttatttttttggtttttcgagacagggcttctctgtatagctctggctgtcctggaactcactttgtagaccaggctggcctcgaactcaggaatctgtctgcctcccgagtgctgggattaaattaaaggcgtgcgccaccacgctcggcttccatgatcttaatgtgctgtagaatgcatgcacgtccaaaagctctactcttactgctacattttaggaatttgaggacccaagggatgcagatgatgctgtttatgaacttgatgggaaggaactttgcagtgaaaggtaagcatgattggtttataagtagttctcccagggaagtagagcagagtagacatttgcatatgtaagtgctctcatgtgtagtgtgtgaatgctttttttacctctttgtagggtgacgattgaacatgcccgggctcggtctcgaggtggaagaggtagaggacgatactccgaccgttttagcagtcgcagacctcgaaatgatagacggtatgtggtttgggtttggctgttacggttgggttagggtccgttagcattaaatggttctcagactagttaagtctagttagtccacatgctgagaacccagaaacttgatggaatcactatcttggcttaaacttgtcacatagcatttgacattgtgacataacatctgacactgaatgcTTATTTTTCTTTCTTCAGAAATGCTCCACCTGTAAGAACAGAAAATCGACTTATAGTTGAGAATTTATCCTCAAGAGTCAGCTGGCAGGTTTGTTGAAATACAGTTTTGAGATATTTTTTAATGTGGCATTTAAAAGTGTTAATGGGTAGTTAAT
This mutation is a 694 bp deletion beginning at Chromosome 12 position 80,947,084 bp and ending after 80,947,777 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
41158 | GCT GAG AAC CCA GAA ACT TGA | Wild type Forward | A | |||
41159 | CAG GTG GAG CAT TTC TGA AG | Common | A | |||
41160 | CTG GCT GGA GTT GAT TCT CA | Mutant Forward | A | |||
41161 | Fluorophore-1 | TGG CTT AAA CTT GTC ACA TAG CA | Quencher-1 | WT Probe | ||
42553 | Fluorophore-2 | CAT CCC TAG CTT AGG GTT ATA GTA TCT G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
41158 | 0.40 uM |
41159 | 0.40 uM |
41160 | 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 |