Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr6:87825430-87825537 108bp GAGCAGGCTTGTGTGTCAGA TAACCTCGATTTCCTGGGACT
Mut = 115 bp
Wt = 108 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
CATTTTTTTAAGCTTACTTTTTTGTGGTGCTGGGAATCGAGCCAGGGCTACTTGCATGCTAGACAAACACTACTGAGCCACATCTCTCTAGGGTCACTTTTAAgggttttgaaaacagtgcacaaatgttcttctatatagagtgtttttcatgtgtgaccttattatctcaaattgtattgtgaacaaaagagcaggcttgtgtgtcagatctattcttcctctttctttcccaacaagaaagttggccccaagatgctggatcatgaaggtaaagaagtcccaggaaatcgaggttacaagtactttggggcagcaaaagatttgcctggtgtcagagagctatttgagaaagaacgtaagtagttcatgtgtttgagagtgcgttggcctttaattgttgctactgagtgagagaagtatgctgtatgctatctggtaggaattattgctcccagattttaaaaaatagtttaaaatttgtattcctaagtttattctatatttttgtagcatggtctcatgtacccccagGCTTTGAATGCCTCATCTTCTTATATCCACCTGTTAGGAATATAGACATCTATCCTTTTTGTTAAAAAACAAAAAACAAAAAACAAAAAAACCATTGTTCACGCTGGCCTAAGACTTGCCAACTAACTTCTGTCTCAGCCTTCATAG
This mutation is a 431 bp deletion beginning at Chromosome 6 position 87,825,195 bp and ending after 87,825,625 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
51347 | GAG CAG GCT TGT GTG TCA GA | Wild type Forward | A | |||
51348 | TAA CCT CGA TTT CCT GGG ACT | Wild type Reverse | A | |||
51349 | GAG CCA GGG CTA CTT GCA T | Mutant Forward | A | |||
51350 | GAT GTC TAT ATT CCT AAC AGG TGG A | Mutant Reverse | A | |||
51351 | Fluorophore-1 | AGT TGG CCC CAA GAT GCT | Quencher-1 | WT Probe | ||
51352 | Fluorophore-2 | CCA CAT CTC TCT AGG GTC ACT TTT AAG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
51347 | 0.40 uM |
51348 | 0.40 uM |
51349 | 0.40 uM |
51350 | 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 |