Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr5:145095934+145096041 108bp AGAAGGAGGGGAAGAAACCA AGCACAGGTCACTTGCTACG
Mutant= 93 bp
Wild Type = 108 bp
wt sequence, deletion in lower case
CCTGGGCTTCTTAACACCGTGTGCCAAAAGAAAACACAAATTCCCAGTTCTGTTCTACCTGGCTTTGTAAGGATCCTGCGTCCTCTGGTGTCCTTAGGAGAGAAACAGCCTTGGTTTTGTATGAGATAATAACCATAGATAGGAGAAGGAGGGGAAGAAACCACTAAGCCCAcagttaccccatgcccagctgtcacccgccaatagcccagtcccttgccactggccaggcgtagcaagtgacctgtgcttcacttttaggtattgactgggctcctaagagcgaccgtattgtcacttgtggggcagaccgcaacgcctatgtctggagtcagaaagatggcatctggaagcccaccctggtgatcctgaggattaaccgtgcagccacttttgtgaagtggtccccacttgagaacaagtttgctgtggggagcggagcccggctcatctctgtctgttactttgagtctgagaatgactggtgagccaagttgcatgtgtcctctcactctgctttgtatactccatgctgggatgctgtgctcctgggacctgttgaggctctgtgtactgcagtgcccttcctggcctggagaggctttctccctctcaaCTCGTATGTGCATGACTCAGCTCCCATACCTTTTCTTTGACTCTTCACTTCCCTTCCTTACTCCTCTACTTCCTTTTGTCCATTACTTCCTCCCTACCCCTTCTTTCCCTTCCCTCCTTCAGCTCCTTCATTTCCTCCTTCCAGCTCCCGACTTATCTTAATTCACATGGGCAACA
A 444 bp deletion beginning at Chromosome 5 position 145,095,963 bp and ending after 145,096,406 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
45117 | AGA AGG AGG GGA AGA AAC CA | Common | A | |||
45118 | AGC ACA GGT CAC TTG CTA CG | Wild type Reverse | A | |||
45119 | GGA GTA AGG AAG GGA AGT GAA GA | Mutant Reverse | A | |||
45120 | Fluorophore-1 | TTA CCC CAT GCC CAG CT | Quencher-1 | WT Probe | ||
45121 | Fluorophore-2 | TGC ATG ACT CAG CTC CCA TAC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
45117 | 0.40 uM |
45118 | 0.40 uM |
45119 | 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 |