Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr9:45900465-45900569 105bp GCACTTAGTATAGGACACACTCAAAAC TTCTCCACCATCAGAACAAACTTCT
Mut= 90 bp
Wt= 105 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
ATCTAAATATTTATGAAAATATGCATAACTAAGTTAGTATATGTAACTACCGTAACTAACATTGTGCTATTGTTTCCTATTCACAAAAGCTGTTGGCAGTGGGCATTTATTTTTAGATCTTGAGGGTCACTTGTTTTTAAGCACTTAGTATAGGACACACTCAAAACTTTTGCAAGGTCTCACTGACCAGGTTAgaatacctacaaattgttgggtagaaagaagtttgttctgatggtggagaaggcctatagcatctaggtttgttctagtggctgtgttggggatgtggctcactagtagagcactgtccagcatgggtttgatccccagcactggtgctagtagcagaatcctgggtctttggtgtgctagtcaggtgacataccactgagctacaacctcatcccagattttttttttttttttttttttttttttttttttggatagccccaaagactctgcccagaaacagaagaacttgtctccgccgagtgtaagtagccaaatgataaccaaggagagtaacagaaatgcccatttggaacatccagaacagaatcctggttcatcagtgggtgatacttcagcagcgcacgaggaagttgtaggagaaaacttggtagccacagccctttgtctttctggcaatgggtctcagtctgatttgaaggacttgaccaacccagcaggagaggagggggacacgagcctccgggagagcctccatccagtcactcggtctcttaaggcagggtgccactcaaaacaacttgcctccgggaattgctctgaagagaaatgcccagcagcctccgtcctaaaggaaggtagcagggatgcaggcttggacttgctacctgtagtaccgcctgcaaatggggttgaaggagtgagagtggatcaggatgatgatcaggatagctcttccttgaagctttctcagaacattgctgtgcaggtcaagtatcacatttctactactaaaatggaattggggacgggaggggagaagccatttaggtggtagagaactgcattgtgtcattttttgctagtagtcatgacacagtttgcatatagtatcatgtaatggcttcattctgctattgcagaaacaagaaagccagtatctaattgtataatatgcaggaattattatgacctatgtgtaacctctttgGATGAAAACTTGATTGGTGTAAATCCTTCCTGTTCTTTCCTTTTATTTTTTTCTTTTTCTTTTCCTTTTCCTTTTTCCTTTTTCCTTTTTCCTTTTTCCTCTCCTCTCCTCTCCTCTCCTCTCCTCTCCTCTCCTCTCCTCTCCTCTCCTCTCCTCTCCTCTCCTCTCCTCTTCTCTTCTCT
This mutation is a 992 bp deletion beginning at Chromosome 9 position 45,899,524 bp and ending after 45,900,515 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
40632 | GCA CTT AGT ATA GGA CAC ACT CAA AAC | Common | A | |||
40633 | TTC TCC ACC ATC AGA ACA AAC TTC T | Wild type Reverse | A | |||
40634 | AGA ACA GGA AGG ATT TAC ACC A | Mutant Reverse | A | |||
40635 | Fluorophore-1 | AAT ACC TAC AAA TTG TTG GGT AGA A | Quencher-1 | WT Probe | ||
40636 | Fluorophore-2 | CAC TGA CCA GGT TAG ATG AAA ACT T | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
40632 | 0.40 uM |
40633 | 0.40 uM |
40634 | 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 |