Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr6:47827130-47827240 111bp TCTCTCCAGCCCACAAGTCA CGAAAGACCATCTCATTGTTCA
Mut= 118 bp
Wt= 111 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case, and insertion with carrots ^g^ (A insertion):
GTCTGTATATCACATCTGTGCAATGCCTCTGGAGAGCAGACAAGGGGGCACTAGGTTCTCTAGGACTAGAGTCAGTGATGGCTGTGAGCTACCATCCGAGAGTTGGGCATGCTGAGCCATCTCTCCAGCCCACAAGTCACTTTTGCAGAAGAGTCTGCTGAGTTAAAAACAG^ctgtccactctgtgagtctcaaattacttatgagagtgaacaatgagatggtctttcgtagccgtcatgacggtatctgtatgatgtctttcacttcagttcccactgacttttgaagatgtagccatttacttctctgagcaagagtggcaacacttagaggcctggcagaaggaactttacaagcaagtcatgagaaccaattacgagactctcatctctctaggtaaggagctttcaacttctctggtgattcttgcaactggatcttctcagagccccttggtcacttgatacacctgttaggtagcaagattgcttagatataacccagaaaggctttttcatctctttattgttgttgttaatactaatgtatacatcaccggtttgtccttagaggt^GGACCTTACAGGGAAGTATTTAAGACAGCATGTAGTATCATAGTCAGAGGTAGCAAGGATGAATGTTAAAACTTTCCCCATCACACTTTACTCTGAGATTGCCTGATTTTTCCACTGAGAAGCACACCTGCCTGTGCCCATAAAGTACCTGTATGCCTTAACCTAGAAAGTGGCTTCTCTGCA
This mutation is a 404 bp deletion beginning at Chromosome 6 position 47,826,784 bp and ending after 47,827,187 bp (GRCm38/mm10). In addition, there is a single bp (A) insertion at the deletion site.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46812 | TCT CTC CAG CCC ACA AGT CA | Common | A | |||
46813 | CGA AAG ACC ATC TCA TTG TTC A | Wild type Reverse | A | |||
46814 | CAT TCA TCC TTG CTA CCT CTG | Mutant Reverse | A | |||
46815 | Fluorophore-1 | CTG TCC ACT CTG TGA GTC TCA AAT | Quencher-1 | WT Probe | ||
46817 | Fluorophore-2 | AAA CAG AGG ACC TTA CAG GGA AG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
46812 | 0.40 uM |
46813 | 0.40 uM |
46814 | 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 |