Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr9:111333275+111333369 95bp GATATGGTGTGTATGTGGAATGG TGATGATGGCATGAAGATGG
Mut= 100 bp
Wt= 95 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TTTGACCCACACAATGGGTACAATACCCTGGAGTTCCCTGCTTATAGAGCCTGCCTCCAAGCCTGCAGAGCCCATTTCTACAGCCAGCTTCCAGGAGACTGTAATCATGGATCCTGAGAGGATATGGTGTGTATGTGGAATGGGCCATGCCACATCTGCATTTTTTGAAGccttggcaggaatgccatcccatccccatcttcatgccatcatcagctttacgttttctccagtgtaattttacatttcctctctacaaatataaaaatatttgtattgattggtgtgggatggaattaagatgtcgaaaccatttgtcaatttaaaaagtgtgagcagaatatttgggagcaagaagcaggtttgtacattgtaaagggccccgtccttttagggggttaagaggacagattttatatgcatatcagagatttcctgctaaccgctctgcctggtgacagcggtcagttccagagtcaggcccttgaggactgtgcctttgcagaatgatggctttattggtggctggtttgttcttcacaggactcctcctcgggaactggctatgctgctgtgcaacaaatccaatgcattttacaacctcgggaaatggaacgaggcttttcttgctgccaaggagtgtctccaatgggacccaacctatgtgaaggtatgggagggtggctgcttccctcttcctcctcttgatttgattgcttttttttttcctccttgtcaaagtggatattctagaaatgcttccccttgaacgcactTGCTCTTCTGAGGTCCATGGGCTGGGGGAGCCTTCCCTCCAGAGGAGCTAAGAGCAGGATAGTGAGTGTTGACACATGCTGCTGGGAGAGTGGGGCCTGACTGCCTGGTAGCTGGAGCTCAGTGAGAGGCGGGAGGAGCTCACTTAGACCTGAAGTAGTTAGACACAGATG
This mutation is a 606 bp deletion beginning at Chromosome 9 position 111,333,325 bp and ending after 111,333,930 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
40821 | GAT ATG GTG TGT ATG TGG AAT GG | Common | A | |||
40822 | TGA TGA TGG CAT GAA GAT GG | Wild type Reverse | A | |||
40823 | TAG CTC CTC TGG AGG GAA GG | Mutant Reverse | A | |||
40824 | Fluorophore-1 | CTT GGC AGG AAT GCC ATC | Quencher-1 | WT Probe | ||
40825 | Fluorophore-2 | TTG AAG TGC TCT TCT GAG GTC C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
40821 | 0.40 uM |
40822 | 0.40 uM |
40823 | 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 |