Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 123 bp
Wild Type = 123 bp
chr5:34178963-34179085 123bp CTTCAGCCTTGAGGTAAATCC AATCCCACAGAACTAGGCTCA
Wt Sequence (deletions in lower case): CAGGCTGCTCCGGAGGGGAAATATTATCTAGGTCAGGAGGGAATTCCTGGAGAGGGATCTAACTACTTCAGCCTTGAGGTAAATCCCCAGGCCCAAAGGCAAGCCTACAGCAGCGcagaggcccatggagggtctggctggccacttcccaccttgcaccagacacatgagcctagttctgtgggatttgctgcctagcttgatagtctatctctggctctttcagacgagctaaactcaggctctacttggagcagctcaagcagctggtacccctgggccctgacagcacacgtcacactactctgagccttctgaagcgtgctaagatgcacatcaaggtaagtctcctcagctaccctgatgccccccagccatccccacccttgGGCAGCATTGGTTCAGAGGACCTGGAGGTGGGTGGTGGAGCCACCTAGCAGGTACGTGTCAGGAGCTTATGGGGAGGCCCAATGGGAGCTG
This is a 274 bp deletion beginning at Chromosome 5 position 34,178,762 bp and ending after 34,179,035 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
60338 | CTT CAG CCT TGA GGT AAA TCC | Common | A | |||
60339 | AAT CCC ACA GAA CTA GGC TCA | Wild type Reverse | A | |||
60340 | CCC ATA AGC TCC TGA CAC G | Mutant Reverse | A | |||
60341 | Fluorophore-1 | AGC AGC GGG CAG CAT TG | Quencher-1 | MUT Probe | ||
60342 | Fluorophore-2 | TCT GGC TGG CCA CTT CCC | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
60338 | 0.40 uM |
60339 | 0.40 uM |
60340 | 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 |