Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr1:106995614+106995732 119bp ATGGCTTGCATCTGACTTGG TCTGGTTCAGAAGGATCTGACT
Mut= 86 bp
Wt= 119 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletions in lower case):
CAGAAATATCCAAGTTTTAGTGGAGAAGGATTATCTGGGGAATTATTTACCCTTGTCAAATATTTAGCATCTATAAATAGATAAAACAAATGCAGGAAACACTTAGTATGCAGTTACATTTCAGACCCAAAGTGCAAAATGGATCAGAAAAGACAGATCTGTGGTATTGGGGCTGGTGAAGAGAGCGGCTTTATGCAGCTGAGATTGGTTATGGCTTGCATCTGACTTGGCAGAAAGTCAAATGGCAGTGGGGACCAATGgacatctctattcttccttcagatttgttccccagaggaaggattgtagtcagatccttctgaaccagaatctagacttgcctctgcccagataacccactgaggtttttgtcttgattttgaagatcgagaagagagaagaaatacatcatcaactccaaatgctgttgactgaaataagcaaattttctaacgattatgacctgatcataagcaacaggctgttcggagaaaaaacatacctcttccttcaagtaagttttgcagtgtttgccacatctatatgtggtaccaagaaatggccagtatccctgttaggactgagttctggggctatgctgccatgttggctgcttgccatgggactatttcactagtcattctagtctgctttccagacaagcagttcctttcttagtcttctgagtctgtatctgtttttttctttaattgtatctgcctgagcatgcatggaagaggtaatgagaaacatttttctcggatctaaaagtccaggttttgagattagttctcctaacgaacagacacatttagttaaaaagcttttgtgcttaataagtaaatccttccatctctttctatttctttcctaaagaaatacatagattatgttgaaaaatattaccatgcatctctggagcctgttgattttgtaaatgctgcagacgaaagtcgaaagaagattaattcctgggttgaaagccaaacaaatggtagagtatgaattgggttttcaccaagaaacaccttttgaacattggctgtgaccaaagccttgggcttgatgcaacacagatgccagatggttgagctttgggagaagctcgagatgggtttggcagaattgaacagtcaagcatcagcataaagctcatctagggtgccgcagagggtagaatggcctctgCATTCGTGGAGAGATGCAACAGTGGGGAATTGTTCCCACAGGCTGTGGCTCACACTACCTTTGCAGTCTTAGGGATGGTGCCATGGTTACCTTTAGCTTATTCACTGTACCACAGACTTTCTTTCTTTCTGAGTCTGGGGCCCTCAGTGCAAATGGCATAGAGGGTTTCCACTTTCTTTATTAAGAGAAGTTCATACAACTCTTGTGCCAGGACCCATCTGCTGCCTACAGCTAGGGTCACTTGGGTCTCTTTCACACTTGGGAATCT
This mutation is a 928 bp deletion beginning at Chromosome 1 position 106,995,664 bp and ending after 106,996,591 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
64380 | ATG GCT TGC ATC TGA CTT GG | Common | A | |||
64381 | TCT GGT TCA GAA GGA TCT GAC T | Wild type Reverse | A | |||
64382 | GGA ACA ATT CCC CAC TGT TG | Mutant Reverse | A | |||
64383 | Fluorophore-1 | CTT CCT TCA GAT TTG TTC CCC A | Quencher-1 | WT Probe | ||
64384 | Fluorophore-2 | ACC AAT GCA TTC GTG GAG AGA T | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
64380 | 0.40 uM |
64381 | 0.40 uM |
64382 | 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 |