Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr3:95137426+95137520 95bp AAACCCTATAGCAGCCTCCA CTCAAACATTTCTGGCTTAGTATTCC
Mut = 107 bp
Wt = 95 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
TTGGTAACTCAGTTAATAAAAGTGCTTGCCACACACACATAGTAAACCCTATAGCAGCCTCCAGGCTCTACCCccaagtacactgttttcaaccacacacaccaataagtttggaatactaagccagaaatgtttgagaacatctgctctacttagtgaaaaggaagaaaaagagcttgctatgttttatgaggttggtggattttactgtcagacattaatttcgttgcttttgtttgaagatggaacagattaagcgtacaaaccgcctttatactaacgactccatcttcctgaagaaaacgctctacatccccatcctatcagagcccagagacttgtttaatggtttggattctgaggaggaaaatgatggagaggaagaggtgcgcccgagtaaggatgagatcggatcatcttcggggaggaggaagaatcgagggtcagggtcgggtcggcccaatggcacaggcctcccgcctcaccaggaaacctccacccccagccatgacctttctgcctctgatttccttaagaagcttgactcacagatcagcctgtcaaagaaggctgctgcccagaagctgaggaaaggggagagtgggtgagtctctggccgctccttctagctccctgcagagaccctcttcttttagctggtcacaaactaagaagtaaccaaagtacCCGAGGACAGGCAAGTGGATGCCTTTCTCCCCATGTGGTCCCCAAATCATGAAAGAGCCAGTGGCATCTGGAATGGGGTAGTGGGTGGGAAGCAGGGTCTGTGGGGTCTTACGCCAGTGGTAGCGC
This mutation is a 614 bp deletion beginning at Chromosome 3 position 95,137,456 bp and ending after 95,138,069 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
51025 | AAA CCC TAT AGC AGC CTC CA | Common | A | |||
51026 | CTC AAA CAT TTC TGG CTT AGT ATT CC | Wild type Reverse | A | |||
51027 | CCC ATT CCA GAT GCC ACT | Mutant Reverse | A | |||
51028 | Fluorophore-1 | CAC TGT TTT CAA CCA CAC ACA CC | Quencher-1 | WT Probe | ||
51029 | Fluorophore-2 | CTA CCC CCG AGG ACA GGC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
51025 | 0.40 uM |
51026 | 0.40 uM |
51027 | 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 |