Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr14:14119788-14119885 98bp CTGCAGATGAGAGGTGCTC CAGAAAGATTCTCTTATCCAGTGAG
Mut= 113 bp
Wt= 98 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case and insertions with carrots ^g^ (G insertion):
TTGTCGCGGCTGGACTCCCGGCAAAGGCGCCGCGGCTTTAGTGATGCCTGTCGGTGTTTAGCTGCCGGCCAAGGGACAGTCAGGGTTCTGTCCCTAGTAGTCGAGTCGAATCGGTGTCCTTTCCCTGACATGAGGACTCCTTTGGAACAGGAGCCGACCAGAGCAAACTGAGGATTCGATTCCCT^tccccttggccttagtcgcgatcagacctgctaagatcggctctcaagcaggctcccagcttcaaaccttagccgtgctacccaggcccatagttgggaggcgtctgctagctgaccttcggtggggtgggaaaagcgagcagaagctaacaggatacctcataatgcgtgatttgtggcttttagatatggctccttattatgaagccttgtgtaaatccctcgactggcagatggacgtggatctcctgagtaaaatgaagaaagcaaacgaagaagagttgaaacgtttggatgaggagctggaggatgcagagaagaatctgggagaaagtgaaattcgagatgcaatgatggcaaaagcagagtacctctgtcagataggtgacaaggtcagtgagatgccctgcccgctctgggtaggtgacagggtcagtgagatgccctgcccgctctgggtaggtgacagggtcagtgagatgccctgcccgctctgcagatgagaggtgctccctattttctggaggtacagaccaatgttgtacatc^AGTGCAAATGAGTAATATCTCACTGGATAAGAGAATCTTTCTGTTTTAACTGGAAGGATAATCATATTTACCTGTACTGAAGAGTCAGTACAGGAACAGTAGATACATGACAATGTTGCAGCACCCAATTCCCTGCTCTTAACAGAGCAAGGTGTGCGTTAGTCTTCCTT
This mutation is a 548 bp deletion beginning at Chromosome 14 position 14,119,831 bp and ending after 14,120,378 bp (GRCm38/mm10). In addition, there is a single bp (G) insertion at the deletion site that will not alter the results of the exon deletion.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
41220 | CTG CAG ATG AGA GGT GCT C | Wild type Forward | A | |||
41221 | CAG AAA GAT TCT CTT ATC CAG TGA G | Common | A | |||
41222 | TCC TTT CCC TGA CAT GAG GAC | Mutant Forward | A | |||
41223 | Fluorophore-1 | CTG GAG GTA CAG ACC AAT GTT GTA | Quencher-1 | WT Probe | ||
41224 | Fluorophore-2 | TCG ATT CCC TGA GTG CAA A | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
41220 | 0.40 uM |
41221 | 0.40 uM |
41222 | 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 |