Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr18:61009177-61009277 101bp CTTTGAATTCCCAGCCTGATAC TTCCACCTACCATAATAGGCAAC
Mut = 108 bp
Wt = 101 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
TTCTGATGCCTTCCTCAGGGAGCCCACATTCTCTGAAATCTTTTGCTTCCTGTGCCAAGGGTACCCTGGCTTCTTCCCTCTGTCCCCCTTGGAGCCTCTTGTCCTGAACACCTTCCAGACTGGCCACCTGTCCTCCGGTAAGCTCCACACTGTAGGCAAAGACTGAGCCATTAGCATTAGGAGTTCTGATGCCCCGCCTGGTTCTCCTGGCCAAGCAATCAAAACCCTCAGGTCGTTCATGACCCCTCCTGGAGCCTCTGCTTTCTTCACACTTGGAATAGAGTGTGGGTGAGTAGTTGGCTCAGGTGTAGAATGTTTACTGCTATAATCACTAACACGTGGTGACAACACACAGTGACTGCCACAAAGATCTTCACTGGCAGTGATATTAAAGTGGgtcatccttggcctatttctgctagccaagatggagacgggcagcttcctaaggcctgagccagctgccggcccctaaggatctgcaagctaggccctcttgagtccaggtgaaatcaccggatcatctgaacctgatgcgagatttcccactcaaccttgctcaacccacggtctcagtttggcctcaccctcctccctctctgctttttttcttctctctctctctcttactggcagcccatcaccccagacctgctgatgactcccagtgaccagggcgatgtggacctggatgtagacttcgcagcagacaggggcaactggacgggcaagctggacttcctgctgtcctgcatcggctactgcgtgggcttgggaaatgtctggcgctttccctaccgagcctacaccaatggaggcggtatgggcctgggaacctcttgggagtagctggggagacagaccaaggcttgggggctataggatggcctttggagtgaccttaggttagagggaggtggcttactctgcctgtcccttttcaccgcctaagcccctcctcctgctttgaattcccagcctgatacccaggtttcctccctaagacctaaagctttcctagaacctatagtggGCAGACATTGTTGCCTATTATGGTAGGTGGAAAGGGTAGTACCCACTGTCACTATTCATTGGCTTTGCAGTGTATCCTGTCCCCTCCCCCACCTCTCCCAGCGCCCTGCTCTACCCACAGTTCCCTTTCTAGAATCTTCCTTGTTCCCTCCTCTGCTGATTCTTTCCATTCAGACTGAAAAACGCAAGATCT
This mutation is a 636 bp deletion beginning at Chromosome 18 position 61,009,209 bp and ending after 61,009,844 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48905 | TTC CAC CTA CCA TAA TAG GCA AC | Common | A | |||
48906 | CTT TGA ATT CCC AGC CTG ATA C | Wild type Forward | A | |||
48907 | GCT ATA ATC ACT AAC ACG TGG TGA C | Mutant Forward | A | |||
48908 | Fluorophore-1 | CAG GTT TCC TCC CTA AGA CCT AA | Quencher-1 | WT Probe | ||
48909 | Fluorophore-2 | CAG TGA TAT TAA AGT GGG CAG ACA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48905 | 0.40 uM |
48906 | 0.40 uM |
48907 | 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 |