Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 76bp
Wild Type = 89 bp
>chr17:74747859+74747947 89bp ATTGTATGGCTTGTGAAGGT GGAGAAGGAGCTACAAACTC
Wt Sequence (deletion in lower case):
TTCAGGAGGCTGAGGCTGGTGGACCTTTGTGAGTTTAAGACTAGCTTGGTTTGCCTAGTGAGTTCAGGCCAGCCAGGGCCACATAGTGAGACCCTGTTTTAAATATCAACAAGAGACAGAACACCAAGCCAAACTAAACAAAAACTCCAAAGAAAATCAAAATAAAAATAATGTACCCCAACCTCCCTCCAAAAACCCCAGCaagcatcaatatcatttccattagcagtctattgaattaatgaatgaataaatgaatgaaagaatgaaagaatgaaatgtaactcttgttctaaatattttcttttgtgtcccactagaatcttgagctcaatgatgatactgttctgaatgaaataaagttagcagatagtgaacggtttcagatgccagacctgtgtgcggaggagcttgccgtggtccttggagtctggtacgttaatgtctgacttggctgtgttgaacgctttgactgttgtcccctgagtttgggaagatcttcatcaaaacctccttgtacagtaggtattgtatggcttgtgaaggtggagacttcttgggcaacgatttaaaggaTCAGAGGTTCAGAATCTATTGAGTTTGTAGCTCCTTCTCCTAGTCTTTGGTTATCTTCAACTAGGTGTAGATTTTTAATCAAGTCCTAATAGACGAGAGTGATTGTGTGTGGGGGATGCATGGAGAGGCAGAGGTGGTCTGTGGATGTTCACACCAGTGCTCCCCACCTTATCTTTCGTTAGACAGGATTTCTCACTGAACCTGATGCTCACCTCTGTGTTGCCTATAAAAGGGATCCCTTCTTTGTCCTCATAAGACTAGGTTACAGAT
This mutation is a 374 bp deletion of region Chr17:74,747,534-74,747,907 (GRCm38/mm10)
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
71604 | ATT GTA TGG CTT GTG AAG GT | Wild type Forward | A | |||
71605 | GGA GAA GGA GCT ACA AAC TC | Common | A | |||
71606 | AAT AAT GTA CCC CAA CCT CC | Mutant Forward | A | |||
71607 | Fluorophore-1 | TTG GGC AAC GAT TTA AAG GA | Quencher-1 | WT Probe | ||
71608 | Fluorophore-2 | AAC CCC AGC TCA GAG GTT CA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
71604 | 0.40 uM |
71605 | 0.40 uM |
71606 | 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 |