Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr7:35794645-35794735 91bp TGTCATTGCTATTGGGGACA AGATGAAGACGGTGGGTCAG
Mutant= 133 bp
Wild Type = 91 bp
Wt sequence with deleted region in lower case
CAAAGTACTTCCCAAAATTCAGTATTCCTTATAGTAGGATGTGAATATATTTTCAGAATTTCAAAAATTTGAGCAGAGAAGTGTGATTTTTCGTTATCTACAGTTAAAATTTGGCATAAGAAATAGTTTATCCAGAGTTACATGAAATCATGTTGGCTTCACAGACTGATGCTTTCCAGTAGCTCGAGGTGACTCCACAGGCCCCAAGGATGGCTGTTTCTTTAAACAAAGAATCATGTCTCTTTAGTTCTAGAATCTCAAGGGTTTCTTTAAACAAAGAGTCATGTCTCTTTAGTTCTAGAATTGCAAGGGATTCaagggacattagttctagaatctcaaggaggctgggtgacaggataaggtgttaaagtcttacccaaggaacagggtctttatctgtctctaggagaccgtttcaaaaaggaattctgagctgtttaagtctattagggacagttgggtgcattgataggaacctcccaacttagaagatatttaaacatttcagtcagacaccagagttgacattatcccataacccatgctctctgctgtgcactgtctctagatatggaagaaagcagcagcattgccatgttggtgcaagagattggagaaccggaggctgtgttaactgctgaaggagtcctcagcccttcctcagaagttgaccaacaaagaaaagctaaagcagatccattggtccacgttatccagaagctaagcaagatagtggggcatgagaagtcacaaaaatgtctactgattgggaaaaagcgcccacgtccaagtgagacagcaaactctcttgaaaagctagagaactgtgagattccagctaaagccactgagtcccctgctgctggggttaggaaaaccgagatgtcacaggcaagttccactcttgcttcaaatgatgggaaggctatgtcctaccagtgtagcctttgtaagttcctgtcaccatccttttctgtgctgaaagagcacgtcaagcagcatgggcagcagcatgacgtgatgctcatgtgctcggagtgccacgccacctccaggagccagcaggagctcgaagcgcacgtggtgagtgagcacgagaacagtgccagtagccaggcccggtccagcccctccggtcagggagccacagaaagaaagagtgagaccatggtagacatcccagtgaacatgggcagtccacagacccatgctgtccagagtgcagccatggcagagtcgggtcggaggaagtggtatgcatatgaacagtatggcatgtaccggtgcttattctgtagctacacctgcggccagcagcggatgttaaagacacatgcttggaaacatgcaggggaagttaactgctcctacccaatctttgagaatgaaaatgagccccttggccttctggcttcttctatgtctgctgcacctggtggggttgatgcagttgtcattgctattggggacagcgagctgagcatccacaatgggccatctgtacaagtacagatttgcagctctgacccaccgtcttcatctcctttagaacaaagcacagaggagggagtacacctaaaccaggcagtcacgctggacgctaacgaggaagaaatgctggaggtgatgtctgattccgaggagaatctctttgctgatagtttgctatcttcagcacagaaaatcattagcagtagcccaaacaaaaaaggtcacgtgaatgtcattgtggaacgtttgccaagtgctgaagaaaccctcccaccaaaacatttcctcattaatgccgaaatggaagaggggaagagcccgagcccctccgaagcccagactgggtgtgtaggagcaggaaatatgtaccatgctgataaatgtactgttgatattggggggttgatcatcggctggagcagtgcagagaagaaagacagtgagctaagtaagggcctggctcctgatgagaatgccccaccaggacggagaaggacaaattctgagtctctcaggctgcactccttagctgcagaagcccttgtcaccatgccgataagagctgctgaactaacaagagccagccttgggcactatggggacataaaccttttagatccagacactggacaaaggcaggtcagtggtccattggcaacatactcaaaaaaaatcatgtctcctcttaagaattctacagatggagtaacaagttttaaccaaagcaattccactgtggtagctctcccagagggaaggcaggaattgtcagatgggcaagttaagacaggcatcagtatgtcccttcttaccgtaatagaaaaactgagagaaaggacagaccagaatgcatcagatgatgacattttgaaagagttgcaggacaatgcccagtgtcagcccaacagcgatgggagtttgttggggtccaacgtggtggagtacatcccagatgccgagcggccctaccgatgccgcctgtgcaactactcaagtggcaacaggggctacatcaagcagcacctgcgtgtccatcggcagaggcagccttaccagtgtcctatctgtgagcacatagctgagaacagcaaagacttggaaagccacatgatcaaccactgtaaaaccagaatacaccaatgtaagcagtgcaaagaatctttccattacaaggtgagcggcggtctcaagggttggcagggactttatagctgtagtccagccaccccagtttgtttatcaatgaaaatgagttccagagaagtttggtaatttgcccaaaggtacccccaacaaattaccattgaagataggtctggaagctaagtctccatggttgctgtccagagctttcccttagtctgatgccgtgccctttgcagcctcagcgcctgttgctttcATGTTTTCCTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGAAGCACGGGTTTTCACTTAGAAGGGAACATAGCGTAGACTGACAGGCACGTGTCTGACGGCAAAGGCTAACTGTGCATCTGCACACAGACCAGTATTCCCTGCACTCATAGCAGCTGCTACACAACCAGCAAACAGTGGTCTCTCACTGCAGAAGGGAAGCAGCCTGTGAGACCTGCTAGGAGGCAGCACAAGAGAAATGATACCATATTTGCTATTGCTGGACACTTTGAGGGACTTTGTTAGGAACCAACTCCAAGACCTCACACCAGCTAGGCAAATGCTTGCCTACCTGTGAAGTGCATTCCCAGCCCTTGTTTTTGTGAGAGTCCTCCTGAATGTCCCAGGCAGAA
A 2580 bp deletion beginning at Chromosome 7 position 35,793,294 bp and ending after 35,795,873 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
45148 | TGT CAT TGC TAT TGG GGA CA | Wild type Forward | A | |||
45149 | AGA TGA AGA CGG TGG GTC AG | Wild type Reverse | A | |||
45150 | Fluorophore-1 | TCC ACA ATG GGC CAT CTG | Quencher-1 | WT Probe | ||
46376 | TTG CAA GGG ATT CAT GTT TTC | Mutant Forward | A | |||
46377 | TAG CCT TTG CCG TCA GAC AC | Mutant Reverse | A | |||
47215 | Fluorophore-2 | CAC GGG TTT TCA CTT AGA AGG GA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
45148 | 0.40 uM |
45149 | 0.40 uM |
46376 | 0.40 uM |
46377 | 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 |