Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr8:8683447-8683604 158bp ATTTTTTAGCCAGAAGTGCTGA CCCCTACCCTCCCAAAAA
Mut= 124 bp
Wt= 158 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case, insertion in ^g^ (ACCTG Insertion):
GATTTTTATCTCCATTTGAATTTCTGTATTACTTATCCTGGCTGCAGTTTTTTATCCATTCATTTATTTATTTATTTTAGTGTGGTGTGAAAACAAGCTATATAATTATTAAGGCTATAAGAAATGAGACTCAGTTTAGTAGTAGGTTTAAAGTTAGTT^ttctaattcttaggttctttaaaatattaagttgtcttttctacttttatttgctagtcggcagcaggaaatagaagaaaaactcattgaggaagaaacagcacgaagagtggaagaattggtagcaaaaagggtagaagaagaattggagaaaaggaaggatgaaattgagcgagaagttctccgaagggttgaagaagccaaacgcatcatggaaaagcagttgctcgaagaactcgagcgacagagacaagctgagcttgcagcacaaaaagccagagaggtaacgctcggtcgtttggaaagtagagacagtccatggcaaaactttcagtgtcaggtgtgcctcctgctcagtccagaacgagatggaatgcgactatcttaattcctttctcatctaaacttacatggctgcgaaagataattttttagccagaagtgctgactggtacttaaaagttactttcttaaaacttattcaaggatatttttgatccgatggaactggcttacatatttgagaagtgtttgaaacttttgc^CATGGCTGCAGGACTACATTCTTTTTTGGGAGGGTAGGGGGAACCAGGGAGTGGTAAAGGGAAAAGGCTAAAGATCCACCCGCGGTTGCATTTTCTTCTCTGTTATCACTCTGCTACCTAGACTGTGAGAGGCTTTGCCTTCAGTCAGATTACAGAGAGCAGGGT
The mutation is a 544 bp deletion beginning at Chromosome 8 position 8,683,487 bp and ending after 8,684,030 bp (GRCm38/mm10). In addition, there is a 5 bp insertion (ACCTG) at the deletion site.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
45186 | Fluorophore-1 | CTG GCT TAC ATA TTT GAG AAG TGT | Quencher-1 | WT Probe | ||
45187 | Fluorophore-2 | AGG TTT AAA GTT AGT TAC CTG CAT GGC | Quencher-2 | MUT Probe | ||
47420 | ATT TTT TAG CCA GAA GTG CTG A | Wild type Forward | A | |||
47423 | CCC CTA CCC TCC CAA AAA | Common | A | |||
47424 | TGT GGT GTG AAA ACA AGC TAT | Mutant Forward | A |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
47420 | 0.40 uM |
47423 | 0.40 uM |
47424 | 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 |