Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr10:122448339+122448466 128bp CGCTGCATGACTTTAAATTGG CAGGGGTGGAGCTGAGAAT
Mut= 129 bp
Wt= 128 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GGGCCTGGGGGCTTGGAAGGGTGGACCCCAAATCCAGGCGCACTGAGTTCGCAGCAGCAGCAGACGCGGATCTCTACGTACGCTTTTACTACACCTGTTGACGCGCTGCATGACTTTAAATTGGCTTATAAAGGAGGCTGGAGAAAttccgaggagcccagagaagtttggggcagatccggaaagcgcccggctctgcagaggaatctaggattctcagctccacccctgcagcaccaaagagaaaaggacaaatcaccaacgacttggaggggttgggtgtaggggacagtctccagccactaagatgagaaggagcgcatcctgaggcagcctaagactgctttgggagcagggagagtccgctcccttgcctcggacaagccggagcagggacacgcaagaacatcccatccaaaaccactctgagcgcgcgtgggattgcgcagcgaagagagcgaggtaaggaaggacggtgcgcagaacttaggacgcgggtcttcgggtcctccgtggagaagaggaagcagcccacgaggaacagggttccccggctctgtgccgacagcatgagtttcccgcgaggctcccacgatctgcccgcgggcaactccagcccgtggtggcctctgaccaccgagggcgccaacagcagccgggaagcagctgggctcggggaaggcggcagcccgccgggggacgtacgcaacgaggagctggcgaagctggaggttaccgtgctggcggtgattttcgtggtggccgtgctgggtaatagcagtgtgctgctggcgctgcatcgcacgccacgcaagacatcccgcatgcatctcttcatccgacacctcagcctggcagacctggcggtcgccttcttccaagtgttaccacagctgtgctgggacatcacctaccgcttccgcgggccggactggctgtgccgggtggtgaagcacttgcaggtgtttgccatgttcgcatcttcctacatgctggtggtgatgacggctgaccgctacatcgccgtgtgccacccgctcaagaccctgcagcagcccgcgcgccgctcgcgcctcatgatcgccgcctcttgggggctgagtttcgttctgagcataccacagtactttatcttctctgtgatcgaatttgaggtgaacaatggcaccaaagcccaagattgctgggctaccttcatcccgccctggggtacccgtgcctacgtgacctggatgaccagcggggtcttcgtggtacccgtgatcatcttgggtacctgctatggcttcatctgttaccacatctggcgcaatgtccgagggaagacagcatcgcgacagagcaaaggcggcaagggctctggggaagccgcgggtcccttccacaaggggcttctggtcacgccttgtgtcagcagcgtgaagagcatttcccgtgccaagatccgcacagtgaagatgacctttgtgattgtaagcgcctacatcctctgctggacacctttcttcatcgtccagatgtggtcagtctgggataccaatttcgtttggaccggtatgtactgagactactatggaagaggtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgcctgtgtgtttgtggtgtgtgttggggggggagtgtttgtggtgtgtgtgtgtatgtatgttatttttcatacaacctattcaaTAGTTAAGCTTCTATGTAAGAAGTCTTAAATGAACAAAATTGCAAATTCCTGGGACCTCCACCCCAGGTACACTGGAGCCCAGGATTCAGCATCTGCAGCATGTGTGACCACCCTAAGGCCACCAGCTGTGCCTAGAGTCACTAGTTACAAAACACTTGTGACATTTTGCCAAATTAC
This mutation is a 1550 bp deletion beginning at Chromosome 10 position 122448382 bp and ending after 122449931 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
43365 | CGC TGC ATG ACT TTA AAT TGG | Common | A | |||
43366 | CAG GGG TGG AGC TGA GAA T | Wild type Reverse | A | |||
43367 | ATC CTG GGC TCC AGT GTA CC | Mutant Reverse | A | |||
43368 | Fluorophore-1 | CGA GGA GCC CAG AGA AGT TT | Quencher-1 | WT Probe | ||
43369 | Fluorophore-2 | CAA ATT CCT GGG ACC TCC A | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
43365 | 0.40 uM |
43366 | 0.40 uM |
43367 | 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 |