Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 83 bp
Wild Type = 110 bp
chr1:106000997+106001106 110bp CCTCCAGACGGACTCACTGT GCAGCAAGAGCAACAACTTC
Wt Sequence (deletions in lower case):
AGTAGATAAACTTTATGTAATGATTAGCATACACAGTACCTCGGGAAATTAACATAAAGTATACTAGTTATTTTATATGTCCTTTTGATTCTGGTTCATGTGGTATAGAATGAATACCACTGGTAACTCTGattagcaacattggatttctgtgtgtaagatagctgggaaagaaaatatttgaaaagattattacttttaaataaaaatgagcattgatactgctacttgtgactctgacacatggtatctttttacccttaatgtttaatcacttgtaactagtataatcattattcagactgtatgacttgactctgtggaattttctcctctgatgtagattaacacattttctcttctagaacgactctgcctgtggtgactatatacagagtaatgagactggtttagtagagcaagcccagatacctccagacggactcactgtggcccctcatcgagctcagcgagaaggtgtgctgcatttgttgttttcacactgatcATCATAGGGTTTTGAAGTTGTTGCTCTTGCTGCTGGTGATGATGGTGGGTGGGTGGGTGGTTGGTTTGTTGGTTGAGAAAGGGTCTCACATAGCCCAAGTTAGCCCAGATTCACTATATATCTAAAGCTGAC
This is a 377 bp deletion beginning at Chromosome 1 position 105,928,427 bp and ending after 105,928,803 bp (GRCm39/mm39).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
59125 | CCT CCA GAC GGA CTC ACT GT | Wild type Forward | A | |||
59126 | GCA GCA AGA GCA ACA ACT TC | Common | A | |||
59127 | CTT TTG ATT CTG GTT CAT GTG G | Mutant Forward | A | |||
59128 | Fluorophore-1 | ACC ACT GGT AAC TCT GAT CAT AGG G | Quencher-1 | MUT Probe | ||
59129 | Fluorophore-2 | AGC TCA GCG AGA AGG TGT GC | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
59125 | 0.40 uM |
59126 | 0.40 uM |
59127 | 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 |