Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr2:181308518-181308634 117bp CTGAGCAATTGGAATGAGATG ATTCAACCTGCCCTCCCTCT
Mut= 120 bp
Wt= 117 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
AGATGGAAGCAGACAGATAAAGAATTCAAAGTCATTGTAGGCTACAAAGTGAATTTAAGGCCAGTCGGATTACAGGAGACCCCTTCACAACACAAAACAAATATGCACATAGATAAAGGCCACTGTAGAATACAAAGACCTAGAAATACcacaggatcatggtacctgctcaccttgactttacatccacagcctacaaggagaagatgaaggagttatctgtgctgtcgctcatttgctcctgcttctactctcagcctcacccgaacaccatctaccagtatggaggtgagtgtatccacacagctgctggccctgctcagctagggcctgacaaccttacttctctcgtttcttctggcctctggcaatgctgtgaactggctggagagagtcattctgggaggaggggcctatgctaaagagtaacggcaccccttcttaacccttgttgggatgtgtgctggaaataaacctctcatcctgcctgtatgcccaaaccaggcctcctgaagtcccatctggtcttgtcctacagatatggaagtgaagcagctggataagcgggcctctggccagagcttcgaggtcatcctcaagtctccttctgacctatctccagagagccctgtgctctcttctcctcccaagaggaaggatgcttccttggaagagctgcagaagcggctggaggcagctgaggagcggaggaaggcaaggactctccttacacacttgacattgctgaccaggccctgtgcttggtcagggcattgggctgggtagtgcagaacttactgctgagcaattggaatgagatgaaagatggatgtgccaagatggtaacaaacacagggcaataggaacCACATTCTTGAGGGGGACCTCGATCATGGCAGAGGGAGGGCAGGTTGAATTCCCCCAGAAAGAGCCCTTAAGACATAAAGCTGAGGCTAGTGCTCCTTTCAGGACAGAAGATCGAGGAGAAAAGGTCTCACATCTAAAGACAGTTAGCTGGGTTGGGTA
This mutation is a 718 bp deletion beginning at Chromosome 2 position 181,308,568 bp and ending after 181,309,285 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
47011 | CTG AGC AAT TGG AAT GAG ATG | Wild type Forward | A | |||
47012 | ATT CAA CCT GCC CTC CCT CT | Common | A | |||
47013 | CCC CTT CAC AAC ACA AAA CA | Mutant Forward | A | |||
47014 | Fluorophore-1 | ATG GAT GTG CCA AGA TGG TAA | Quencher-1 | WT Probe | ||
47015 | Fluorophore-2 | AGG CCA CTG TAG AAT ACA AAG ACC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
47011 | 0.40 uM |
47012 | 0.40 uM |
47013 | 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 |