Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr18:58582442+58582535 94bp AATAAGAGGAAGGCCACCCAAG CTGACCCTGAAGTGCTCCTG
Mut= 101 bp
Wt= 94 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TGCACATTCAATGCTATAGTAGTACACATGCAATTACTGATTTTGATTTCCATTCGACAAATCACAACAAGTACATTTGCTATCAATACTGCATGACTGCATTAAGACACGGATCTTAAAGGTCATGTTTCCAAATGAGTTTGTAACcagtgaccgaggatgtggcatgaaagcctggatctgttaggacatcactatcagctgtgtgccacgttaacttaatgccttttatcaatctcttcacataggcgccacgtgtgtgctaaagaagaaattctccgcaagccagttttggaatgattgcaagaagtacaacgtgactgtgtttcagtacattggggaactttgccgttatctgtgcaaacagcctcaggtaagagagtgttgctggagatgctaagggcttcagacagaatatgctttctttgcttttgctgtacaatgctccagaagagaatctatcaaacacacacaggtgttcatagtatacaggatagaagaaatataaaagtccaagaaaataagaggaaggccacccaaggaagtctcaataatacgctttgtcctgcagCAGGGCCAGGCAGATCCATAGTCAGGAGCACTTCAGGGTCAGGCCAATTCCAGTTTGATTGTGAAATTAGATGCATAGATTTACAGAAAAACAATTCCAAGTTGAACGCAGATAATGCCTTCTATATAAACCTG
This mutation is a 424 bp deletion beginning at Chromosome 18 position 58,582,070 bp and ending after 58,582,493 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44695 | AAT AAG AGG AAG GCC ACC CAA G | Wild type Forward | A | |||
44696 | CTG ACC CTG AAG TGC TCC TG | Common | A | |||
44697 | CTG CAT GAC TGC ATT AAG ACA C | Mutant Forward | A | |||
44698 | Fluorophore-1 | CAA TAA TAC GCT TTG TCC TGC A | Quencher-1 | WT Probe | ||
44699 | Fluorophore-2 | AAT GAG TTT GTA ACC AGG GCC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44695 | 0.40 uM |
44696 | 0.40 uM |
44697 | 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 |