Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr3:137885308+137885444 137bp GCTGGAAGTAGCACGCATTG CTGAGCACGCCCTCTACTTG
Mut= 138 bp
Wt= 137 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case; and insertions with carrots ^g^ (TTCATAGC & T insertions):
GAGGGTTTTACTGTGTAGCTCTGGCTTTCCTGTAGTTCACAAAGATCTGTCTGTCCAGCTCTGACAAGCAACCTGTTACTTAAAGAGACATTATGAAGCCTGAACTGGTGGCTTGTGTTTGTAATCCCGGCACCGGGAAACTAAAGCAGAGTTGAGAGTTCAAAGCCGGATTGGGCCACGTAATTCTAGGTCAGCCCCAACTGCATGGTAAGGTCATGCCTAAACAAAGGGAAAAACATAACAACAATCTCCCCTCCCTAATAAAAAAGATAAAGGAGGACGAGGCCATGT^ttagtctctccagaagagctcatcttgtctgctttgtcttttttccggagccctttgtctctgtgtcctgatgtgagtaggttgaagtgtcctttatctctctttacacagcgttattggaaataattatgaaaaacggcagcaccgctggaagtagcacgcattgccgaaaacctcctgggagcagcgaccagagcaagcccagctgtggaaaggacggcacttctggtgctggggaaggcgggaaagtctataccaaagaccaagtagagggcgtgctcaggtaggtgtgaatgcgagtgtgtccgcaccatgatcgttgactgtgattggacccaatcgacgttataatgcctaaaataaaaatgtatttatatatagcacagtttaatagag^GGAAATGAAGG^AGTTACCTGGGTGCCCTGTCCTTCATTATTTTGAAGTCAGCCTGGAGGGAGTTAACTTTCTGGAAGTTTCCACTCATTTAAATTAGCTATAAGTCACAGTGAGTTTTTAAAATAATGATCTCAGCTTTGAGTCTATGCACTGTTTGGACTGTAGTATTGAATGGATTGCTTTGTTGTGATACGTAGTTGAAAATAGGAGAATTTCATATGCGGGAAGGCT
This mutation is a 396 bp deletion beginning at Chromosome 3 position 137,885,162 bp and ending after 137,885,557 bp (GRCm38/mm10). In addition, there is an 8 bp insertion (TTCATAGC) at the deletion site and a single bp insertion (T) 11 bp before the deletion site.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48011 | GCT GGA AGT AGC ACG CAT TG | Wild type Forward | A | |||
48012 | CTG AGC ACG CCC TCT ACT TG | Wild type Reverse | A | |||
48013 | TCT AGG TCA GCC CCA ACT GC | Mutant Forward | A | |||
48014 | CCC AGG TAA CTT CCT TCA TTT C | Mutant Reverse | A | |||
48015 | Fluorophore-1 | CTC CTG GGA GCA GCG AC | Quencher-1 | WT Probe | ||
48016 | Fluorophore-2 | CGA GGC CAT GTG CTA TGA A | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48011 | 0.40 uM |
48012 | 0.40 uM |
48013 | 0.40 uM |
48014 | 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 |