Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr9:104238232-104238312 81bp GAAGAAGTTGTGTGTGTGAGAGC AAACAGTAACATGTGAAAGTCATCC
Mut= 87 bp
Wt= 81 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GAATTGGAGTTACACAGACAGTTGTGAGCTGCCACATGGGTGCTAGGAACTGAACACAGGTCCTCTGAAAGAGCACACAGTGCTCTTAAACCACTGAGCTATCTTTCTAGCCCCAAGGTCATACTTTTTATGCAAGAATATTAATAGTTCCCACTAAAAtaagtggcggatagattttaattttgataacattttaggacattaaatttgaaaatataaaaataaactatatttaatcaccattgaactattgatcagccaacatatttctatttatagaaaaatatgaaagtgtattcattttgtgttaatatcatatgttttcatttaaaaatttgttgcttaatactaaatgtaagcatgtctttccttgtaggtataaacgagtcttttcagttggaacccatgcagttactacctataatcccaataccttagaagtaacaaatcaggtaattttaatagaagttgttgagggtttatgtgaacctttaaaagtcaagattgcagcccatcttccctgctgaagtcagcagttcagaagagaaatgtcagcctgaagatgaatgtgtatatatgatgccttgtgttcagacctagaagtgaagaagttgtgtgtgtgagagcgagcgcgggcacatgctttctagactcTCGAGGGGATGACTTTCACATGTTACTGTTTTTAGCCATCTCTATAAAATTCGTCCCTTTCTAAAGTTGTTACTATTAATCAAGCATATTAGTTATGTCACAAGTGTTATTGTTCACAGACTTTCCAAAGAACTAGAATTACTTGTTTGTAAAGGCAA
This mutation is a 495 bp deletion beginning at Chromosome 9 position 104,238,263 bp and ending after 104,238,757 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
40349 | GAA GAA GTT GTG TGT GTG AGA GC | Wild type Forward | A | |||
40350 | AAA CAG TAA CAT GTG AAA GTC ATC C | Common | A | |||
40351 | TTT CTA GCC CCA AGG TCA TAC | Mutant Forward | A | |||
40352 | Fluorophore-1 | CGG GCA CAT GCT TTC TAG AC | Quencher-1 | WT Probe | ||
40353 | Fluorophore-2 | ATA TTA ATA GTT CCC ACT AAA ATC GAG G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
40349 | 0.40 uM |
40350 | 0.40 uM |
40351 | 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 |