Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr13:76056727-76056836 110bp TCCGGATTAAGGATGGAAGA CCTTGCTCAGAAGTCAGGTTG
Mutant= 90 bp
Wild Type = 110 bp
Wt Sequence: TAACTTTGGGAAATCGCCGAGACCCAGCGGTGTGGCCCGCGCCACCTCTCATAGACTTTGGCCTTTTAAGCGGTATCTCTCTTTCCTCCGGATTAAGGATGGAAGATCctttcagcctcgcaattctgaatcccgcatccaacctttctgtacccactcagccaagctggagtctcaacctgacttctgagcaaggagcctctgtcccaggaccgcattcgcctccacgcgggccacccagccaccggatccatctggtcttcttggggatcatcctggtggccgcggtagctggcaacaccactgtgctctgccgcctgtgcggcggtagcagcggaccctggccgggtcccaagcgtcgcaagatggacttcctgcttgtgcagctagcagcggctgacctgtacgcaagtggaggaacagcattgtcgcagctggcttgggagctgctgggcgacccgagaccggctctgggcgacttggcttgtcgcctttcccatctgctgcaggcatcggggcggggcgcctccgcccacctggtggcgctcattgccttggagcgccagctcgcggtgcgcattcctcagggccctcagttgccagcacgtgccctggctgccctgagctggctgctggctctgctactggcgctgcctccgaccttcgtggtgcgctgggatgctcctccatcttcaaccgccaatgcctggccagggaagcattgctgcaggggcatcttcgcgcccctgccgcgctggcacctgcaggtctacgcgctctatgaagctatcgtgggcttcgcggcgccggtcgctctcttaggcttctcttgcggccacctgctgtgtgtctggtggcagcgtgggtctcaggctccggtggctaggatgccctggtcgcccagtatggccagagcctccttgcccagcgcgttgccccaagccaaggtgcaaagcttgaaaatgagcctggcgctggcactgctcttcgtgggctgcgacctgccctacttcgccgctcgcttggcagccgcctggtcatcgaagcccgcgggtgactgggagagagagagcctggtggcggcgatgcgcgtcctagaggtggccaacagcgccatcaaccctttgatctaccttttcttccaggcgggcgactgccgtctctggcggaggctgcggaggcgcctaggagttctgtgctgcgtgcgggaggaagaagccgatatcagcgaatgggctggggaccaccaggcactccaccgccatcgctggccccaccctcattatcaccatgcccggcgggaggagcggaaccagggctgcctgcgcccacccccgccacgccccagaccgccgccctgctcctgtgaaagtgcCTTCTAGGAACTGCTGAACGGACAGGTCCTTGGCCACTGTAGCACAGCCTTCAG
Mutant Sequence: ctttcagcctcgcaattctgaatcccgcatccaacctttctgtacccactcagccaagctggagtctcaacctgacttctgagcaaggagcctctgtcccaggaccgcattcgcctccacgcgggccacccagccaccggatccatctggtcttcttggggatcatcctggtggccgcggtagctggcaacaccactgtgctctgccgcctgtgcggcggtagcagcggaccctggccgggtcccaagcgtcgcaagatggacttcctgcttgtgcagctagcagcggctgacctgtacgcaagtggaggaacagcattgtcgcagctggcttgggagctgctgggcgacccgagaccggctctgggcgacttggcttgtcgcctttcccatctgctgcaggcatcggggcggggcgcctccgcccacctggtggcgctcattgccttggagcgccagctcgcggtgcgcattcctcagggccctcagttgccagcacgtgccctggctgccctgagctggctgctggctctgctactggcgctgcctccgaccttcgtggtgcgctgggatgctcctccatcttcaaccgccaatgcctggccagggaagcattgctgcaggggcatcttcgcgcccctgccgcgctggcacctgcaggtctacgcgctctatgaagctatcgtgggcttcgcggcgccggtcgctctcttaggcttctcttgcggccacctgctgtgtgtctggtggcagcgtgggtctcaggctccggtggctaggatgccctggtcgcccagtatggccagagcctccttgcccagcgcgttgccccaagccaaggtgcaaagcttgaaaatgagcctggcgctggcactgctcttcgtgggctgcgacctgccctacttcgccgctcgcttggcagccgcctggtcatcgaagcccgcgggtgactgggagagagagagcctggtggcggcgatgcgcgtcctagaggtggccaacagcgccatcaaccctttgatctaccttttcttccaggcgggcgactgccgtctctggcggaggctgcggaggcgcctaggagttctgtgctgcgtgcgggaggaagaagccgatatcagcgaatgggctggggaccaccaggcactccaccgccatcgctggccccaccctcattatcaccatgcccggcgggaggagcggaaccagggctgcctgcgcccacccccgccacgccccagaccgccgccctgctcctgtgaaagtgc
1267 bp deletion beginning at Chromosome 13 negative strand position 76,056,814 bp and ending after 76,055,548 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
37622 | TCC GGA TTA AGG ATG GAA GA | Common | A | |||
37623 | CCT TGC TCA GAA GTC AGG TTG | Wild type Reverse | A | |||
37624 | ACA CCC TCC GTT GTC TGA AG | Mutant Reverse | A | |||
37625 | Fluorophore-1 | CTG AAT CCC GCA TCC AAC | Quencher-1 | WT Probe | ||
37626 | Fluorophore-2 | TGC TGA ACG GAC AGG TCC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
37622 | 0.40 uM |
37623 | 0.40 uM |
37624 | 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 |