Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr15:84693392+84693500 109bp CTTCCCCGGATGTTTCTAGC AGTGAAGAAGGACCCCAGCTC
Mut= 115 bp
Wt= 109 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case; and insertion with carrots ^g^ (A insertion):
GTTTGGTCGCATTGGGACCCTCAGACTTCTCTTCTGTGAGATGCATAGTGAGCCAGCCACTCTGTGGGTCACAGAGGGAAAATAATCTCTTGGGCCACTTGTATTGCTGTGCTGAGCAGGTTGGTCCCTGGCTTCCCCGGATGTTTCTAGCCCTCACCAG^tactgtaggatctcaacttgtccccatctgccctcccccacaggcagctgctgaagacggagctggggtccttcttcactgagtacctgcaggtacgtgggccttgccacagaggaaggcttgctctgtctctcaaaagaggtctctatagttctgtgacctgtgacagcccgggcaggctctaggctggcctctcggggtgggtggggttttgactgcttggcaggagggtgggtctcatgtcttggctgcttgttggcgggctcctggtgggtcctgtaagccaaaccctcctgaaagagctacaggtcagtgatggcgcctctgttggctttcagaaccagctgctcacgaagggcatggtgattcttcgtgacaagattcgcttctacgaaggtgagtcttactcggagctgagtgtggccagcctggctgtgatgtggtgtggttgatgttctgggacagtggcgtggcagatgct^GGGAGGCAACGGCGGCTCTCTGGTTAAGATGGCCGGAAAGGAAGGGAACAGCATTGGGCAGCGGGACTGAGGTCTGGACTGGGAGGGGACCTTGGAGAGAGTCTGCACCCACCCCCTTGGGAGCCCAGGAGGGCTGCGCAACCTAGGGAG
This mutation is a 481 bp deletion beginning at Chromosome 15 position 84,693,421 bp and ending after 84,693,901 bp (GRCm38/mm10). There is also a single bp (A) insertion at the deletion site.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44021 | CTT CCC CGG ATG TTT CTA GC | Common | A | |||
44022 | AGT GAA GAA GGA CCC CAG CTC | Wild type Reverse | A | |||
44023 | CTC CCA GTC CAG ACC TCA GT | Mutant Reverse | A | |||
44024 | Fluorophore-1 | TGT AGG ATC TCA ACT TGT CCC C | Quencher-1 | WT Probe | ||
44025 | Fluorophore-2 | AGA GGG AGG CAA CGG C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44021 | 0.40 uM |
44022 | 0.40 uM |
44023 | 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 |