Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr2:69736202+69736293 92bp GGGTTTAGATGTGTCCTGTGC ACCGTAAGTGAGGCTGTGCT
Mutant= 105 bp
Wild Type = 92 bp
Wt Sequence (deletions in lower case):
CAATACAACAGAACATCATACAACCAGGAATATTTAATAAAAACCTAGTGACAAGTAGAAATACAAGTGGATAGAAATAACTCTAAATGTCAACTGCATGCACTCACTGTCTTCAGCAgtcaataggtatggcttttatattccgaacaagcttttgtaaaactcgtatgggaaaccctttgcactcatgaggttctgctatataagggtatcacatgatttcttattttcagtacactttttttgtttttatagaagctaactttgtttttctgtttttattttatctgatgaatcccaaaagaacaacaaaaccaactcctcatttagatgggtaaagattattttaatatcttacaacatactttattttgttgtattagagtaagaagcttttatttaataatttttacatgacattttaggcatcatgttgtttttgggcaagtgatctctggtcaggaagttgtgagagagattgaaaaccagaaaacagatgccgctagcaaaccatttgctgaagttcggatactcagttgcggagagctgattccaaagtctaagggtaagaacgggtgcacagttaagggaactcttacttggataagtagagattataaataagcatgaggttttgtgggtttagatgtgtcctgtgctgtcagttttttctaatttatatgtgcttctgtacgtatgtGTCTGGGTCAAGCACAGCCTCACTTACGGTTCTTCAGAAGGCATGGACCATATACTGTAAGTACAATATACAATAAGAAAATTATAGTTGTGAAGAACTTTGT
This is a 582 bp deletion beginning at Chromosome 2 position 69,735,682 bp and ending after 69,736,263 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
47620 | CCT AGT GAC AAG TAG AAA TAC AAG TGG | Mutant Forward | A | |||
47621 | GGG TTT AGA TGT GTC CTG TGC | Wild type Forward | A | |||
47622 | ACC GTA AGT GAG GCT GTG CT | Common | A | |||
47623 | Fluorophore-1 | TTT ATA TGT GCT TCT GTA CGT ATG TGT CT | Quencher-1 | WT Probe | ||
47624 | Fluorophore-2 | CAC TCA CTG TCT TCA GCA GTC TG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
47620 | 0.40 uM |
47621 | 0.40 uM |
47622 | 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 |