Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr18:63030212-63030311 100bp CCACGGTCCAGCTTCTCTTC GAGGCACCAGATCAATGAGA
Mut= 99 bp
Wt= 100 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case and insertion with carrots ^g^ (AGAATGGGGACGAGGAGTGTTAGGATATTAATAAAGAACACTATTAGGGAGAGGATTTGAACCTCTGGGAACAAGGTTTTAAGTCTTACGCAATTTCCTGGCTCTGCCACCCTAATAACCTTCTCT insertion):
GGAGCTAAGGTTGGGGTCACCTGTGCATGGTAAGTGGGTCAAGGCTAGTGCTAGTGCTAGCAGTGAGCACTCAGATGTGTTCTAGAAGGTTCTAACAAATTGAGAGGTCATTGTCTCATCCTTACTCTATCTGCCTCACTCTACCTGCCTTGGCTTAGTTACTCATACAAACTCTTTTCACCCGCAAACGTCTCCACTCT^cctaggacactcacaattctgtacaagcttccttagacataaatcccttggctttccaacgtcaagggattgaatgttggaagggacactgtaagaaagacagccagttggcccacccaagatgctagctagcctcagctgtcatcttctcttcttctaacagtgtcatggtctatgggacgaggatgacattgttgacagtaacacagacaaggaaggatcggatgacgagctgtccctcgatcagggcagaagaggctcctcagactcactgaagtctatcaacctggctgcctctgtagagtctgtccacgtgaccttccccgagcagccagcggccatcaggagaaaacgctcctgcagtagctctcagatctccccacggtccagcttctcttcaaataggtccaaaagaggtatctgttgcatggccattaggttga^AATGAGCCCCACATAGTCTCATTGATCTGGTGCCTCTGCATCTGGGGAGGGAAGTGGTTCGGTGCTATCAAAAGACGATGTTGTGACATGGAAGAAACATGCTCTGGCATATGACAGACCCAAGTTCAGAACCTGTTTCATC
This mutation is a 443 bp deletion beginning at Chromosome 18 position 63,030,248 bp and ending after 63,030,690 bp (GRCm38/mm10). In addition, at the deletion site there is a 126 bp insertion (AGAATGGGGACGAGGAGTGTTAGGATATTAATAAAGAACACTATTAGGGAGAGGATTTGAACCTCTGGGAACAAGGTTTTAAGTCTTACGCAATTTCCTGGCTCTGCCACCCTAATAACCTTCTCT) that is from the mouse mitochondrial genome (ChrM 2666-2791).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
42139 | CCA CGG TCC AGC TTC TCT TC | Wild type Forward | A | |||
42140 | GAG GCA CCA GAT CAA TGA GA | Common | A | |||
42142 | Fluorophore-1 | CTG TTG CAT GGC CAT TAG GT | Quencher-1 | WT Probe | ||
43199 | GGT TCA AAT CCT CTC CCT AAT AGT | Mutant Forward | A | |||
43200 | Fluorophore-2 | CCT CGT CCC CAT TCT AAT GA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
42139 | 0.40 uM |
42140 | 0.40 uM |
43199 | 0.40 uM |
Wt Probe | 0.15 uM |
Mutant Probe | 0.15 uM |
DNA |
Step | Temp °C | Time | Note |
---|---|---|---|
1 | 94.0 | -- | |
2 | 94.0 | -- | |
3 | 65.0 | -- | -0.5 C per cycle decrease |
4 | 68.0 | -- | |
5 | -- | repeat steps 2-4 for 10 cycles (Touchdown) | |
6 | 94.0 | -- | |
7 | 60.0 | -- | |
8 | 72.0 | -- | |
9 | -- | repeat steps 6-8 for 28 cycles | |
10 | 72.0 | -- | |
11 | 10.0 | -- | hold |