Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr3:90413194-90413273 80bp GGGGACTGAGAGGGAAATGA GGAGGGAGGAGCGTATGGTA
Mut= 82 bp
Wt= 80 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletions in lower case):
AGAACAATATCATCCTCTCTGGTTTGGGGGGGAGGGCAGCAGTTCTAGAACATTCTTTCTCTCTCAAGGCAAAGGGAAAATGTTTACATCCTAAAagAGAGAACGGTTTATTCTAGCATTCAGGGCAATAGTGGTGGGAGAGTGGACcatcgggatgactgaagagatttattcgattagctgagttgctaattagagctactgtattctgattcttccccctcccaccccttgaaatcaggtggcgatgttacagccaaaaatatctggttggcagaaagtgtcctggatatcctgacggagcagaggtagagtctcccatgaagggtggggctgtgaggggtgtgagtatgggacatgggaaagaaaggctggtggctcagagcactctgaggccaggggaaggtgtggcaggcctgatggtggcagatggagaactaaggtcaaaagatggaggaatgggaaagaaggcgcttgctaaaatatctcagagcccagaaaatctggtctcatctgataagggtttatttttgacccagacctccttggaatgtttatgtttggggggactgagagggaaatgagctcagcagaggggccagccaacCACACGAGCCTGGCAGGTACCATACGCTCCTCCCTCCCAGCCTCAGGCCAGCACATGCTCTTTTTTGGAATGTCACAGGGCCAATGTAGGTGACTAGGGAGCAGAGAATCCTGCTATAAAGTGACATGGGGCCACATTTGAACTTC
This mutation is a 460 bp deletion beginning at Chromosome 3 position 90,413,231 bp and ending after 90,413,690 bp (GRCm38/mm10). In addition, there is a 2 bp deletion (AG) 50 bp before the exon deletion that will not alter the results of the exon deletion.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
40762 | GGG GAC TGA GAG GGA AAT GA | Wild type Forward | A | |||
40763 | GGA GGG AGG AGC GTA TGG TA | Common | A | |||
40764 | ACG GTT TAT TCT AGC ATT CAG G | Mutant Forward | A | |||
40765 | Fluorophore-1 | CAG AGG GGC CAG CCA | Quencher-1 | WT Probe | ||
40766 | Fluorophore-2 | AGT GGA CCA CAC GAG CCT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
40762 | 0.40 uM |
40763 | 0.40 uM |
40764 | 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 |