Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr11:5534128-5534235 108bp CACAGCTGGTGCTGACACCT TTCTGTTCCCTAGTCTCCATGC
Mut= 113 bp
Wt= 108 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TGAAACTTAAACATAAAATAAATGGATGTTTTTTAACTAAATGAGTTTTAGCTGGGCCTGGTAAGCTCTCTCCTCAGTCCCAGCACTCTAGGCAGAAGCAGGCAGAACTCTGAATGAGGCCAGACATAGATACAGAGTGAGACTTTGTCCCAAAAATATATCTTTTTTAATTCGTTAGAGAAATgtcttacatttcgtctataatggattgcttttcttccttccctctcttcccgctgctttcctcctcctaggtgtccagtaagaaagaaacgactgactgaagctgaactcggtgcagttactgatgaatgggctctcggcgcacatcagggaagagaaggtcatggggtaaacacgtgtcctagcagcctttctatgcccagcatgctagatgtggtctgtgaggaaatggatcagacaactggagaaccacagtgtgaagtcgcccgaaggcggctgcaagagattgaggacaggtactgactgcgcgtcacatgtgctgtggtcctgtgtcagctctcctctcctaggctgctcggggtcacgtcctggtgctggagatggggaatgaacaacagacacttagtggtagatggaatagagcttgactaagccttttactgaccgtgtcaagctccgttttcctggacacttcttctaacttgactctattccaaacctttctcggtgaaaaacagcctcatttggccacttttctcagagttgtagcttacatgatctctcgctgcagcacctgtgggtttttcttttagtttcaaattttgtttgttcatttgctacaagaaacacacagtgccttgagtgattttttttttttctttctccaatttattatttaggataattgacgaagatgaagaggttgaaagtgacagaaatgttagccacctcccaagtcttgtcctttctgacaccatgaaaacgggtttgaagagggaatttgatgaagtcttcacaaagaggatgattgagtctatgtaagtgcacagctggtgctgacacctgggaaggctcttgccaagcctgttctcttaaagctgggggggGACAGCAGCAGCCACTCAGGCTCAGCATGGAGACTAGGGAACAGAAGGTGATGTGTCAGATTAGTGACAGTTGACATTGCTATCACGAAGCCCACAAACTAGCACAGCCGTCTGTGGTCATTTTAGTGTTTTAACTCTTGGATTTGTTTTG
This mutation is an 886 bp deletion beginning at Chromosome 11 position 5,534,174 bp and ending after 5,535,059 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44556 | CAC AGC TGG TGC TGA CAC CT | Wild type Forward | A | |||
44557 | TTC TGT TCC CTA GTC TCC ATG C | Common | A | |||
44558 | GGC CAG ACA TAG ATA CAG AGT GAG | Mutant Forward | A | |||
44559 | Fluorophore-1 | AAG GCT CTT GCC AAG CCT | Quencher-1 | WT Probe | ||
44560 | Fluorophore-2 | TTC GTT AGA GAA ATG ACA GCA GC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44556 | 0.40 uM |
44557 | 0.40 uM |
44558 | 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 |