Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr1:14910565-14910688 124bp AGGCCAGAATCCACATCTGA CGTCACTACCACCACCATCA
Mut= 111 bp
Wt= 124 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GTATCTTTTGGGCAATTGCAAGCTAATTATAAATATAAGTCACATGGAACCTAATTATGATTTGCTATAATGTAATCTCAGCATTATAGAACAATTATGAAATTAATATGCAGATTAAAAGTATAGTAACAAGAGGATCTGTTTCTATAAATACTATGTAAAAAATTAACCCAGTGTTTGGATCTATAGGTACTTTCACCTGGTCTCAAGTGGTGTCCATTGGgtaattctgaagctaaaactacatcctatttctatagttcaagaaagtgtacctggttttataagcacttgactccgaggttcggagtgggtagttcttcaggatatattaagtaaatcccaaacaacatttaatgcatattttgttatttctttagtgctgaatataatggatggttatggaaataccccactgcattgtgctgcagaaaaaaatcaagttgaaagtgtaaagtttcttctcagccaaggagcaaatccaaacctccgaaatagaaacatgatgtcaccccttcacatagctgtgcatggcatgtacaacgaagtgatcaaggtgagcctgctggccatcacagtgaggctgactgtggagctaggccagaatccacatctgactcacttactgtttcaaagtgactgacttcaagtgccatgtcatggCAAATGAAACGTAACTATTATGTTTATTTTTCGCCAAATGATGGTGGTGGTAGTGACGATATGACGGCAATGGGGAAGATGATGATGGATAGCACTGATGAGAAAGATGGTGGTAACGACTGTCATAGTAACCAGAGTTAACTTTTCTTGAGTTTTCCTTTATCAGGTATTTTAAAGAAGAGCTGTGTGACTTTATTAAGTTACCATTT
This mutation is a 440 bp deletion beginning at Chromosome 1 position 14,910,623 bp and ending after 14,911,062 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
40826 | AGG CCA GAA TCC ACA TCT GA | Wild type Forward | A | |||
40827 | CGT CAC TAC CAC CAC CAT CA | Common | A | |||
40828 | CCA GTG TTT GGA TCT ATA GGT ACT TTC | Mutant Forward | A | |||
40829 | Fluorophore-1 | CTG TTT CAA AGT GAC TGA CTT CAA GT | Quencher-1 | WT Probe | ||
40830 | Fluorophore-2 | CCA TTG GCA AAT GAA ACG T | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
40826 | 0.40 uM |
40827 | 0.40 uM |
40828 | 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 |