Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr3:116948069-116948166 98bp GCCAAGTCAGTGGGAAAAAC ATTTGCCAGCAATTCCCAAG
Mut= 106 bp
Wt= 98 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletions in lower case, insertion with carrots ^g^ (AT insertion):
CCACCTTATTTTGACAGATTTTTACATGATCATAATAGTTATTTGTAATTATTAAAATATTGATTTGGTTTCTTTTTAAAGAAGAAAGGGGTCAACAAGTTTACAAGGAAGGGAAAATGCCAGATTTATTGAGCCAAGTCAGTGGGAAAAACTAATCTCTGTTGATATG^cccgctgtcgccatgcataagacttgaaagcaataggagatcttgggaattgctggcaaatataagaacatacgtttatattccctgacatcagaaaaaagccctgagggagaaatggctcctggatggaatcggcagtggaaaagaacacgaagagatgaagaagcaaaatcagcaagaccagcaccagacccaggttctagagcaaagcatcctcaggtacggccctcactggggtacttctcaaaccgagctccttccctaaa^AGGGAGCTGGGgtGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGCACACGCATGCTCTCTCAGGTATACTCACACATGTAGAAGCCAGATATGAATGCTGGCATGTCTTCCTCAGTTGCTTCTCCCAGATTTTTAATATTTTTATTAATTCTTGAGAACTCCA
This mutation is a 266 bp deletion beginning at Chromosome 3 position 116,947,864 bp and ending after 116,948,129 bp (GRCm38/mm10).In addition, there is an AT insertion at the deletion site as well as a 2 bp deletion GT 11 bp after the 266 bp deletion that will not alter the results of the deletion.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
40546 | GCC AAG TCA GTG GGA AAA AC | Common | A | |||
40547 | ATT TGC CAG CAA TTC CCA AG | Wild type Reverse | A | |||
40548 | CTG AGA GAG CAT GCG TGT G | Mutant Reverse | A | |||
40549 | Fluorophore-1 | CTG TCG CCA TGC ATA AGA CTT | Quencher-1 | WT Probe | ||
42053 | Fluorophore-2 | ATA TGA TAG GGA GCT GGG GTG T | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
40546 | 0.40 uM |
40547 | 0.40 uM |
40548 | 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 |