Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr7:109557340-109557466 127bp CTTAGGTCTCAGTCAGTCTCTCCAC TGAGGAGCACCTGGGATTTAG
Mut = 145 bp
Wt = 127 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletion in lower case):
TTACTCAGGTCTCTGAGAATGAGCGAGACAGAAAGGTTCGTGGAGGTTGGCCTGCCGTGACTAAAGAGCTATTCTTTACTTGAGGACAGCCTTAAAGATACATATTAAATGAAGGATTTCTTTTCTTTGTTTTGGACATACTAACTCAAGCTGGCCTCAGACTCACTGTGTAGCCAGTGATGACCTTGAACTCACAATCCTCTTGCCTTCATCTCCCAAGTGATGGGATTGGTATGGGAGGGGGCACTTAGAGTATTTTTGAGACTTCTGCAGGAACATAACATCAGAATGAAACACAGTCCCCCTGGTTTTTAGACCACTCACCCTTTTCCTTTTTCCTTTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCTCTTAGGTCTCAGTCAGTCTCTCCACCTCCAGTTTTATACCCACCAagaagtcccatctacccactcagtgacagtgaaacctcagcctgcaggtaccccagccactctaaatcccaggtgctcctcaaggaccggcattctcgaaatccttcactcctaggtcaagatccgtccccagaaacctcaccacccatttgtaccctcaaggctaccagcttcagctatttggacagaaccccttcattacgcaaaagagaggaccagaaggagactgtccagggtgcagtccaggatgtagaaggtgtggctgcttgcctcccccttgcccagagcaccccattcctaggggccgggtctcggagtgtcttgctgagttgcactggtacccgagcccatagcctgggtatccgagagaagatctcagcatgggaaggtcgccgagaggcttcacccaggatgagtctgtgtggagagaagcgggagggccctgggagcgagtggtcggtcagtgagggctgccccagtgtgggctgccccagtgtggtgccatccccctgcagctcagaaaagacctttgatttcaaaggcctccggaggatgagcaggaccttctctgagtgttcctaccccgagacagaggaggaggcagaggcacttcctggacgggactctttataccggctggagaagcgtccaggccggactgagcccagtgccttgctcagggggcatggcatcaggaaagagagctcagcagtgctgagccggatccagaagatcgaacaagccctaaaggagcagcctggccggggtctcccccagctccctagcagctgctacagtgtagaccaagggaggaggaagactggaactttgggcaccttggaggagccaacaggaactgcaagtgtaagccccagcagcagggcaggaggagtggccggagttgctggggaggcaggcccacccctggacagggaaggcagtgcttccatgaagtcagagacccctggaaatagctccagtccccagctgctgcctccgaagagctcccctgatccagctgtgaaccctgttcccaaacccaagcgtacctttgagtacgaggctgacaagaatcctaagactaagccaagcaatggtctacctccttcacccacacctgctgctccaccccccttgccttccaccccagccccaccagtcacccggagacccaagaaggacatgcgtggtcaccggaagtcacagaacaggtaaggCATGTGCTCCTGTGTCAGGAAGCAGGACCTAATGGCATTCTTTTAAATGGAAGAAGCAAGAGCAGCATGTGCTTGGGTCACAGGCGTATGCTACAAGCTCTGAAGCGCTGAGTAATTAATTGTTAGTTTGCCAGTTTGGGCAGAGAAATGGTTCTCCTGCTCCCCACTACCACACACATACCTCCTTTCATTTTTGTGCCTGAACTGGAACCTGCTATAGACCAGGCTGGCCTTAAACTTTTGCCTTGGCCTCAGGGCTGGGTTTAAAGTCATGTACCACCACCAGGCCTGTCCTCCAGCTCTTGAGTGGAAGAAAGGATGAAAGCCACTCTTGCCAGTGCTGGGAGGCTTTGTTAGTGTAGAGCCAGCTATGCTAACAATTC
This mutation is a 1,217 bp deletion beginning at Chromosome 7 position 109,556,205 bp and ending after 109,557,421 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48524 | CTT AGG TCT CAG TCA GTC TCT CCA C | Common | A | |||
48525 | TGA GGA GCA CCT GGG ATT TAG | Wild type Reverse | A | |||
48526 | GAG CTT GTA GCA TAC GCC TGT | Mutant Reverse | A | |||
48527 | Fluorophore-1 | TCA GTG ACA GTG AAA CCT CAG C | Quencher-1 | WT Probe | ||
48528 | Fluorophore-2 | CAC CAC ATG TGC TCC TGT GT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48524 | 0.40 uM |
48525 | 0.40 uM |
48526 | 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 |