Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr8:47527067-47527199 133bp TAACTTCTGGGAGCCCTAAGC AGAAACAAAGCACAGGTGTCAG
Mut= 122 bp
Wt= 133 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
AAGCCAGAACAATTGTTCTTGCCACGTTGAGCATTCTGAGGTCTTTGGAAACTTCTTTCATGAGTGAATGCTTCCATTTTTATTTATGTTTTGGAACAGCCATGTAATATGGTTTGGGCATTATTTGTCCTGTCTATTGTGCTCAGGAGAAAATTCCACACTATTTTCTAGACAAATATGAACATATTAACTTCTGGGAGCCCTAAGCCCTCAGgaagaacaggagccctgctgccagggtgacagacttagaactttattcctgatgacaaacttgaataaatttgactacagagagctgacacctgtgctttgtttctgtttgtcaacaggggaagatgtcattgcttcggagcgggctgcggccttatgcaatgtgtgtgagctctcaggcaagtctctgtttgtgctgccccacactgaccaccttgtgggctacattataaggtaaatcagaggtttttttgttcttaagaaggaaggaaaagttggtaacttagatcctttatttacaaacaacaaagtgttcataaaaacattgtgcatggtgaagttcAGCTTGGCCAAGCACTAAATGAACAGACTGAAAGGAGTCTGAGACTAGCCTGGTCTACATTAGCAAGGTCCACTCTACCTGGGGCTACATAAGTAAGACCCTGAATCAAAAACAAACAGTGAGGATCTTCTAACACTGGCCTGATAGCCAAGCTATCTCTATAGCTACTTAGCCTGTTTTGCTTAAAAGCAGGGAAGAGTTTAATTCCAAAT
This mutation is a 342 bp deletion beginning at Chromosome 8 position 47,526,831 bp and ending after 47,527,172 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
47503 | TAA CTT CTG GGA GCC CTA AGC | Common | A | |||
47504 | AGA AAC AAA GCA CAG GTG TCA G | Wild type Reverse | A | |||
47505 | TAC TTA TGT AGC CCC AGG TAG | Mutant Reverse | A | |||
47506 | Fluorophore-1 | CCT GAT GAC AAA CTT GAA TAA ATT TGA | Quencher-1 | WT Probe | ||
47507 | Fluorophore-2 | CCT GGT CTA CAT TAG CAA GGT CC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
47503 | 0.40 uM |
47504 | 0.40 uM |
47505 | 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 |