Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr2:10085988+10086086 99bp TGTGACATTGTTAGCAGCGTATC CCAACGTGCTTCCCTAAAGC
Mut = 104 bp
Wt = 99 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
GACATGTAGCCTGTCCCCTTCAGACACTGCACATGGCAGTACATAGGCACACATGTGCACAAAACTCCCAAGAGAAGAATTTATCATTAACCCATttgcagggcttaatcgactattgtgtgtaggggggtcggtgatactgtagattagaatttaactccctctttaacatcttttgtagaatggctttaagtgtcactgtatgtctgaatctcatcaaagacaactgttgctggcttcagaaaaccctcagcagtttatggattatttttcagagtaagtaattcaaagacatccttaccttgtaaggttactaagtgggcatgtaatggaagtgttcaatattgtatgcattgtagagcgttgtcccttgtgtgttgagttggctgtaggctcatcattgtgcttggtctttatcttcttttgaaattaaccacagtgtgacattgttagcagcgtatcttagaactttttttcacttcagctctgcctagcctcaaagctctccccaccTAGGGGCTTTAGGGAAGCACGTTGGGATGCGCGCCCTGCGTTT
This mutation is a 418 bp deletion beginning at Chromosome 2 position 10,085,644 bp and ending after 10,086,061 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
51144 | CCA ACG TGC TTC CCT AAA GC | Common | A | |||
51145 | TGT GAC ATT GTT AGC AGC GTA TC | Wild type Forward | A | |||
51146 | CCT TCA GAC ACT GCA CAT GG | Mutant Forward | A | |||
51147 | Fluorophore-1 | CTT CAG CTC TGC CTA GCC TCA | Quencher-1 | WT Probe | ||
51148 | Fluorophore-2 | CAT AGG CAC ACA TGT GCA CAA A | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
51144 | 0.40 uM |
51145 | 0.40 uM |
51146 | 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 |