Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr7:134918843-134918938 96bp ACATAAAGGCCGACCTCACC GAAGCACACATGCAAACGAG
Mut= 93 bp
Wt= 96 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TATCAAACGAAAACCAAGTGTATCCCACATGGAAAAAGTAAGCAGGCTTTAATATCGAGAAGCAGTTCACGCTGTGCCTGCCAGCAACTCCCCGCCTGTCTGATTTTCCTTAACATAAAGGCCGACCTCACCTGTTTAAAAATAATTAGGAAGCCAGCAGctgtgatttggctggacgtccattcggcctcgtttgcatgtgtgcttctgacccactgatgcctctgttgctgtctctgcatgcaggcaccatggtcagcaaggactctggcagatgcatactcacgacaccagagcgggaagtggagcccgccgcctgcctggccctggagatgagatatgccctcgaccctaaccggcagatcaaaaagcgaaacaaagctctgcaggtgcgttttaaggatatctgtgaggcccagaatgagcagagagacacacagctgtcttcgggacctctgggggagaagcgagaggccaaggctgtgtcctgcagggtggcctatcgtaagtatatgacagtgcctgcacgccgatccattcccaatgtcaccaagagcaccggtgtgcagacctcaccagatcttcgcaagtgttaccagacattcccgctggaccgcaagaaaggcagtctcaaaggcctgccggctgcagatgccttcaaaagccagaacaatgggtttctagcagattcaaaagagaagagcgaggcgggacccatggaggagcctcggccttgcagtgcaggaaggattcacaagaccaccgcattggttttccattccaatgagcacgtgaacgcactgggacagccttctggggtcaactgtgcagagctctgtaagtctccagatgtgcttggctacccagaggctgttttgcaaaactctcggcctccctctgaggagcccatccaccagctgcaggggagagctaagcttgacaggggcaccctggactcagaggagccggctccactcacccacggaagggtgtttaaaaccgaggtcgccactgtgtatccgcctgccatcagtaccagggcccctgagcctggcttgtcaaactctgctgctgccagccagtggtctctctgccctgcagatgaggagcagaggcgagccgcccacctcaatgggctacaggcatccacaggctctgctgtggcctgctcaaccccggtgcagtacctgtccccagaatgtagcgaacagcccctgcagaaccagcctagccccacagctgggataggggatgaagaacaccaacaaattgtgcctcacacagaggtggtagacctcaaggcccagctgcaggtgatggagaacttaatcagctctagccaggagaccatcaaagtgctgctgggagttattcaggagctggagaaaggagaagcccatcgggaagggtatgtatggccactgcatcttaccccctgtgggctcaccacttgcaggcccatcagcagccccgatgtcccagccctaaggagtctaatgccggtggtttttgtgtcagggctgcagtctgacaagggtcacagGTGTGTTGAAGGCCAGGCTTCCTGGCACACTCGCCTCTGATACTCATTCGTTGGTTAGCATTTACCCATCTGGCCTTGGGTTTCAATCTCTCCCTGTCTATTCAGGGAGCTGATGGGTCTTCCTTGTGATCTTTTCAGATTGATGTTTTCTA
This mutation is a 1383 bp deletion beginning at Chromosome 7 position 134,917,508 bp and ending after 134,918,890 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
43723 | ACA TAA AGG CCG ACC TCA CC | Common | A | |||
43724 | GAA GCA CAC ATG CAA ACG AG | Wild type Reverse | A | |||
43725 | GAG TAT CAG AGG CGA GTG TGC | Mutant Reverse | A | |||
43726 | Fluorophore-1 | ATT TGG CTG GAC GTC CAT T | Quencher-1 | WT Probe | ||
43727 | Fluorophore-2 | CCA GCA GGT GTG TTG AAG G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
43723 | 0.40 uM |
43724 | 0.40 uM |
43725 | 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 |