Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr3:98427142+98427246 105bp TGCCAACTTGTCTGTTGCAG GGGAACAGAGCCATGTCAG
Mut = 112 bp
Wt = 105 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
GGTTGAGAATCCCCCCTCCCACCCCCGCCTCGTTTTTTTCTTCTGGAAACATATTCCCATCTGGTACAAACCTAGCCATTCGCAGGCTTCAGGTGATTCAGCTGAGCAGAAGCTGGGGTGTCTAGTAGGCGGTGTCTGCCAGGAGAGTTGGCAGTGGGGCAGAGTGCATTATCCTTTAGTCTTAGCAGGAGGAAACTGGTAGGGAAGAGGCCGAGTAAAAAGTAGGGGTAAGGGGGCTGGGGGAGGGGCATTACTCCTTTGGCTCACCCTCCTGGCACATCAGGTCTAGTTGACCCCATTGCCAACTTGTCTGTTGCAGAGGGGCGGCTGAGTgaggaagaaagggctatcctccgtgaggaagaggacgaccagcctggtgtggctgacatggctctgttcccaggactgtcagaatctgacagcatatcccggagtccgcggggagaggaagatgaagaggaggaagacgaagaagaggaaagcgctggggagaaccgcttgatagaggaggaagatccattgcctactcccgtgcttccttggaggcgtcacctctccctgggcggtcgacaccggggtgacaagcctgctcaccgccgcttccaccggctccatcaccccatggccatggacctcggggaactagacagcctgatggccagtatcatggacgcgcccaccatctgcccagactgcggggagagctttagcccgggcgccgcattcctgcagcaccagcgcatccaccgcctggcagaggccgccgccgtggccagcttggagcccttcggtttggccggcgagtgcggaggagtggtggggatgatgggcatgggtatgggcgtgggcatgggcgtggccggcggcttcggggcggggcccacgctggcccggccccctcgcgaaaagcccttccgctgcggggagtgcggcaaaggcttcagccgcaacacctacctgaccaaccacctgcggctgcacacgggcgagcggcccaacctgtgcgccgactgcggcaagagcttcagctggcgcgccgacctgctcaagcaccggcgcctgcacacgggcgagaagccctacccgtgcccggagtgcggcgaagccttcagcctcagctcgcacttgctcagccaccggcgcgcgcacgcggcggcgggcggcggcgcgggctcggcgggctcggcggcggcgctgcggcccttcgcatgcggggagtgcggcaaggggttcgtgcgccgctcgcatctggccaatcaccagcgcatccacaccggcgagaagccgcacggctgcggcgagtgcggcaagcgcttcagctggcgctcggacctggtgaagcaccagcgcgtgcacacgggcgagaagccctacatgtgctccgagtgcggcgagaccttcagcgtgagctcgcacctcttcacgcacaagcgcacgcactcgggcgagcggccctacgtgtgccgggagtgtggcaagggcttcggccgcaactcgcacctcgtcaaccacctgcgcgtgcacaccggcgagaagcccttcggctgcggccagtgcgagaagcgcttcagcgacttctccacgctcacgcagcaccagcgcacgcacaccggcgagaagccctacacatgtctcgagtgcggcaagagcttcatccagagctcccacctcatccgccaccggcgcatccacaccggcaaCAAGCCTCACAAATGCGCGGGCTGTGGCAAGGGCTTCCGCTACAAGACGCACCTTGCACAGCACCAGAAGTTGCACTTGTGTTAGGGAGGGCATCCGAATGAGGCGGCGGTGGGAGTCGTCTGGCTACTGATCTGTAGATGTTGGGGAAGGGGCAGGAGGGACAGTCTGAGAGTGTCTGGGGGAGCCTTTTGGTGTTTGCCACCCCACCCCCCAGGAGATGGTTAATAAGAGTTGTTACTTCGAGGTGCCTACCTCCACCTTCAAAGTAACTCTTAGAAGA
This mutation is a 1360 bp deletion beginning at Chromosome 3 position 98,427,181 bp and ending after 98,428,540 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
49298 | TGC CAA CTT GTC TGT TGC AG | Common | A | |||
49299 | GGG AAC AGA GCC ATG TCA G | Wild type Reverse | A | |||
49300 | AAG TGC AAC TTC TGG TGC TG | Mutant Reverse | A | |||
49301 | Fluorophore-1 | AGG GCT ATC CTC CGT GAG GAA | Quencher-1 | WT Probe | ||
49302 | Fluorophore-2 | AGT CAA GCC TCA CAA ATG CGC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
49298 | 0.40 uM |
49299 | 0.40 uM |
49300 | 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 |