Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr8:23902255+23902354 100bp AGCTACAAGCTTTTTGGGTTAG CATGAACTCATACTCTAAAGGTCCT
Mut= 118 bp
Wt= 100 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case; and insertion with carrots ^g^ (AGCAAG insertion):
GTGAGGCTAAAATTGGCAAAGGCAGAGCTGAATGCCAAATGATAATTTCTGCTAAATAATTCAGGCCATGTGAACTTTGATATTTCCCTTACTTTAATGAGTATAATGTGGGCAATTACCAACTACTCTTCTCACAGAGCCAGACAATGTGCTTTTTCCTAGTTATGGGCTTCATTTTTCTTCGGGTAACTCATATCTGCAAAGTTTGCTATAAATGCAGGGCCTTGCTCATGCGACC^gctgcataaatgtatattactgaggtgatggtaattataacgaggaagtttagcatgtctgtaagctgttcatcaatagacaaatcttatgacatgggacttgagttcttctgtgacactgtctttctccttttccccatgcagggaagcgatgctgacatggtggataagaataaatgctgtacactctgtaacatgtcctttacctcagctgtggtggccgactcacattaccaaggcaaaatccacgccaaaaggttaaaactgttgctaggagagaaacctccactgaaaaccacaggtatgtatggaaagaaaaaaaatgatccaggctggtacagagaacctctcttttcagccataggaatcaacctctttttcagaagctacaagctttttgggttagatggcaggtgccattcaaaacc^CACAGGGAAGGATGGGAAGGCTGAGCCGAACAGGACCTTTAGAGTATGAGTTCATGGGCTTGTGCACTGTCTTTCATCTAATGCTTACAGCCTCCAGGATGTCTGTCACGTTGATTGCATATCATGTGAGATACACATCTCTAGTGCTCTTTCCAAGTTCAGCCCACAGTCCACATTGGTATGGTTGTGCACACTGCATAGGTAGTAGGTTCTTGCCAGTGTCTATGGAAAGCAATTAGCATCCTTGACATTTGTGCTTTGA
This mutation is a 428 bp deletion beginning at Chromosome 8 position 23,901,871 bp and ending after 23,902,298 bp (GRCm38/mm10). In addition, there is a 6 bp insertion (AGCAAG) at the deletion site.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
47844 | AGC TAC AAG CTT TTT GGG TTA G | Wild type Forward | A | |||
47845 | CAT GAA CTC ATA CTC TAA AGG TCC T | Common | A | |||
47846 | CGG GTA ACT CAT ATC TGC AAA GT | Mutant Forward | A | |||
47847 | Fluorophore-1 | ATG GCA GGT GCC ATT CA | Quencher-1 | WT Probe | ||
47848 | Fluorophore-2 | ATG CGA CCC TTG CTC ACA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
47844 | 0.40 uM |
47845 | 0.40 uM |
47846 | 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 |