Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr18:84730298+84730444 147bp AGAACACGCTAGGCTTGGAG GATCTGGAAGCAGGCTGAAG
Mut= 150 bp
Wt= 147 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CTGTTCTGTGCAGATGCAATATTTAATCAATGTTTCCACTAGGCGTTGGCTGGGGAACTCACAGAACAAGGCAATCACCTGAACCCGCCTCCCGGGTGGCCTCCAACACTCTCATGGGCATTTTCTCTCCTCAGCTAATTACAAAGTCCTTTGCCCCCTATTCCTGCCCTgcagtgaccctctgttccttttgttccagtgccaggactatcgccgtggctggctcacgggcgccctgtgcgaggacctgtgtgtgggcggggagctgctgtaccaacgctgcctgtattacgagcgtggtaagaaggtgctgcaggctcagtggcgtggccggaccgttgtcctcaaatccaagagggaggccttctccagcttcccgcccctcactctgctggaggaggaagcaggggcaggtgcccccggcatccctgaggctgagctgctcctgatggtggccggggaggtcaagaacacgctaggcttggagctgcccaacaacagcatagccccgctatggcctgccaggcagggccctggctggcggcaacagctggccagtgcgtggtcactgctccaacaggaggaatacgtatacttcagcctgcttccagatctgagccgccacatcctaccggtgctgggatcctgcggtcacttttatgcagtggagtacttggctgctggtagccctcaccacaaggctctcttcccactggatgatgctggccaagcacaggctattagccacattgccctcagcttcctggacatggtgagccatttcgacagtgacttttcccaccggctccacctctgtgatgtcaaacctgagaacttcgccatcaagagggactttacggtgagtggaagcaggctgccatgtttgtggtaagagaagagattctggtctgtgtaggaggaagtgaattcttggcctctggaaggaagcccatgcaactttgggcagccttgtggttcctggtgattcccgttccagaccacttccttgccaggttgacaaaagggtttaagaaaatgcatctccaacctcaagaggagaggtccagtccagtgaagaagcccacaggaggcagagacaggcagggggcggaggagacatctcacttccttgaggGCAGCTATGAGGTCTAGTTAGTGCTGCTACAGGATCTGTGGTCTTCTGAGGACAGTCTTCCTGGGCTGCAGATGGTGTGCTGTTCTTCCAGGCCAGGCATTCTGTGTCCACAGAACCAGTGTGCAGAGAACAGTCATTGCCAGCCGACATTACTCTTCTGGAGAGATTCT
This mutation is a 965 bp deletion beginning at Chromosome 18 position 84,730,001 bp and ending after 84,730,965 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46077 | AGA ACA CGC TAG GCT TGG AG | Wild type Forward | A | |||
46078 | GAT CTG GAA GCA GGC TGA AG | Wild type Reverse | A | |||
46079 | ACA AGG CAA TCA CCT GAA CC | Mutant Forward | A | |||
46080 | AAG ACC ACA GAT CCT GTA GCA | Mutant Reverse | A | |||
46081 | Fluorophore-1 | CTG GCT GGC GGC AAC | Quencher-1 | WT Probe | ||
46082 | Fluorophore-2 | AGT CCT TTG CCC CCT ATT CC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
46077 | 0.40 uM |
46078 | 0.40 uM |
46079 | 0.40 uM |
46080 | 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 |