Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr18:36788638+36788734 97bp CCGATCGAATTGGGGACT AGTTCAACAAACACACATCACC
Mut= 93 bp
Wt= 97 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case and insertion with carrots ^g^ (CTAC insertion):
AAGGTAAGAAAGAAAACATGCTGTAAATAGATCTTATCTGGGTTTCTGCCCTGCCCTCAAATCCCTGTCATTAGGGAAAGAGTAAGGAAACCATGGCTAGAAGTGGCTATGCATGAGAAGGAAAAAGGCTAAGGAGACATTTTAGAACAAGGTCCGAGTTACACCATGCAGTGGGGACTGTGGCTGGATTTCCTGAGTTGCCTC^gggctttactgaattcactgttagtcccttaagacatccataccaaaggaaaatgagaggtcctgcttcagttagagaaagggcggagctaatgtttggatcttaatgcagatgtcttgggaaggcgtgaggcatgagatggtggcaaagaaaggtctagctcccgaggtggccgatcgaattggggacttcgtccaatatcatggtaagaaccagggtttttag^AGCCATGATAGAACAGGTTGAGGGTGATGTGTGTTTGTTGAACTTCTACCTCTAGATCAGTTCCAGAGTTTAGCCTGGATAAAGGTTCCCTTTTATGTATTCTAACTGCTTTATATCTTCTTAGTAAAGGCAGAAACAGAAGAAGGTCTAGCCATGAGACCAAGGCTGCAGTCCTTTTAAAGAAAATGAGGAAGACTGGCTGGAGCATAT
This mutation is a 225 bp deletion beginning at Chromosome 18 position 36,788,466 bp and ending after 36,788,690 bp (GRCm38/mm10). In addition, there is a 4 bp insertion at the deletion site (CTAC).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
47158 | CCG ATC GAA TTG GGG ACT | Wild type Forward | A | |||
47159 | AGT TCA ACA AAC ACA CAT CAC C | Common | A | |||
47160 | TAC ACC ATG CAG TGG GGA CT | Mutant Forward | A | |||
47161 | Fluorophore-1 | CAA TAT CAT GGT AAG AAC CAG GGT | Quencher-1 | WT Probe | ||
47162 | Fluorophore-2 | CCT GAG TTG CCT CCT ACA GC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
47158 | 0.40 uM |
47159 | 0.40 uM |
47160 | 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 |