Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr10:123059082-123059204 123bp CACGGAAATTTTGGCAGGT ACTCGGGTGCAGGAAGTAGA
Mut= 125 bp
Wt= 123 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GAGGCTTTACAGTGTGCAGTGGGGCGGGGAGGGGGGGGGCGGGGGGGTAGATGTGTACAAAGAGGAGAAGAATTGAGTTCTTCTCCTTGGGTTTTTGCTTGTTCTGTTTTCTGAATACACAGTCTGTACAGTGTTCAGGCATTCCaccgaggtaggccttagagcacaggaaggcggcaggcaaagtgcagtttgcatgtgagtaaccctgatgacggggttctcttaggtgttgtgtgttatgcgttgtgtgttgtgtgtgccactactgggcctctgttgtttgcaaaacaaacatagtagtggcactttgcatttttaattttgtgtttatttctcacacagaatttctgtttatagtaagttttattttctaattatttttgttcctacaggctgctgaatcgggaataataaaagttaaaacaatagctgcaagaaacacggaaattttggcaggtaattttttttgcattgaattcaattagtagtgaagattttactaatagttaaatagactcttaaaaccaccaGTTTTTTGAGCATCTACTTCCTGCACCCGAGTATGGTATTCATACACATGACATAGAACATGTGAGTGCCCCTTAATTGTGAACTGATTTTATTTCCCAGAGGTGAAAGCTGTAAAATTCCTTGAAATGAGAGACCTTAAAGCTACCAGTTCCTTTGAATCAAGGATTTTCTTATTCACCTTTGCTTTC
This mutation is a 388 bp deletion beginning at Chromosome 10 position 123,059,114 bp and ending after 123,059,501 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
41010 | CAC GGA AAT TTT GGC AGG T | Wild type Forward | A | |||
41011 | ACT CGG GTG CAG GAA GTA GA | Common | A | |||
41012 | GTG TAC AAA GAG GAG AAG AAT TGA G | Mutant Forward | A | |||
41013 | Fluorophore-1 | TGC ATT GAA TTC AAT TAG TAG TGA AGA | Quencher-1 | WT Probe | ||
41014 | Fluorophore-2 | CAG GCA TTC CGT TTT TTG A | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
41010 | 0.40 uM |
41011 | 0.40 uM |
41012 | 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 |