Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr7:4528094-4528212 119bp GGAAGTCCCAGCTTCAATCA GAAGGTCTCACTCCGCTCTG
Mut = 113 bp
Wt = 119 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
GTTTTGGTTTGCTTTCAGATAGTTTTATTGTATTCCAGGGGCTATGTCACCCTTTTCTGATCTCTGAAAAATCTTGcccatggtgaacagatgtacacactcaggcacacacacacacacacacacacacacacacacacacacacacacaccgaataaaaagtttaatttattaaaagtatattttaaagacaagacttgagactatatgtcagcttgctttgcttttgggaagtcccagcttcaatcagcagtgctgggtggtggttgcagttcctggattggtgcagtttgtcaagtctccctccctgtttcatccttccctcctccagagcggagtgagaccttcctggagctgtgggggaacgcgctgctgcgaccctcggtagctgccttcctgcgcgctcaggctagccacctggctaatctggtcctggagcctgatcgcctggaggagcaactaccctggcttagtcttcggctgctcaaggtgccacagctgcccacctttgttggagggaagtggaatttgcttgaaatttgggtggctgcaggaagaatggaaatgagcagttcagaggaattgaggggctggagcaggctggcgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtcctaattccacaattccacctctgtaactcccactactgtggcctagGGTTAGGGGAGACTTTGCTCTCTGTGCCTCATTTTTTCCGCCCCCATAATCACTACCACCTAATGGGATGGAAGGGGAGCCCTCAC
This mutation is a 617 bp deletion beginning at Chromosome 7 position 4,527,750 bp and ending after 4,528,366 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
52323 | GGA AGT CCC AGC TTC AAT CA | Wild type Forward | A | |||
52324 | GAA GGT CTC ACT CCG CTC TG | Wild type Reverse | A | |||
52325 | GGG GCT ATG TCA CCC TTT TC | Mutant Forward | A | |||
52326 | CTT CCA TCC CAT TAG GTG GT | Mutant Reverse | A | |||
52327 | Fluorophore-1 | CTG GGT GGT GGT TGC AGT T | Quencher-1 | WT Probe | ||
52328 | Fluorophore-2 | TTG GGT TAG GGG AGA CTT TGC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
52323 | 0.40 uM |
52324 | 0.40 uM |
52325 | 0.40 uM |
52326 | 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 |