Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr16:20608666-20608782 117bp CCTTGGTGCTTGTTTTCTTGA GTCAGCTCCCCAAACCTGA
Mut= 110 bp
Wt= 117 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CACGGCACTGGAGAGGCAGGACAAGGGATCCTTGAAGCAAACTGACTAGCTAGACTCTGTTATATAAATGACAGAGGAAGACACCAGTGTCAACTCCTGACCTTCATATGCATGCACACATAACACACAGACGAAATATATGGGTTCACCCAGAGaatgccgtaggtgtggttacatcagggacgataatgggagaaacctgggaatgaggtagaatctctgggctttggtggtctgaaggcatggggtttagaagtaggcaagttctggactcagaaggaacgagaccatgttatcttcttccagacacagagattgactggaaggcctacatggcccaggtggagggtttcatcaatggcacatatgactacacacagctgcagggtgacaccggacctcttgtgtgagtgaggataggaagtctgaaggcattgtgaagggctgggtgggtaccaggctgatagtcctgccctaggctcatatttcctttcatcttccagctacccagctgggtttttgtacatctttacggggctattctatgctactgaccggggcactgatatccctatggcccagaacatctttgctgtactctaccttgtcaccttggtgcttgttttcttgatataccaccagacctccaaggtgagttgatgccacaggaccaactggccctggcctgaagTGGGTGGTGGGGGTGGCTCAGGTTTGGGGAGCTGACGTTGGTGCCCACCCTTTAGGTACCTCCCTTCGTCTTTTTCTTCATGTGTTGTGCCTCTTACCGTGTCCATTCCATCTTTGTACTGCGGCTCTTCAATGACCCAGTAGCTATGGCCTTGC
This mutation is a 536 bp deletion beginning at Chromosome 16 position 20,608,702 bp and ending after 20,609,237 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
42125 | CCT TGG TGC TTG TTT TCT TGA | Wild type Forward | A | |||
42126 | GTC AGC TCC CCA AAC CTG A | Common | A | |||
42127 | CAC CAG TGT CAA CTC CTG ACC | Mutant Forward | A | |||
42128 | Fluorophore-1 | ATA CCA CCA GAC CTC CAA GGT | Quencher-1 | WT Probe | ||
42129 | Fluorophore-2 | CAC CCA GAG TGG GTG GTG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
42125 | 0.40 uM |
42126 | 0.40 uM |
42127 | 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 |