Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr7:133602348-133602452 105bp GGTTAGCTCCAGAGCAGTACTCA AAACATTTTGCACCCTCATGG
Mutant= 110 bp
Wild Type = 105 bp
Wt sequence with deletion in lower case
TGATGTGAGCAAGGTTAGCTCCAGAGCAGTACTCAGGTTAGTTTTCTGAAAACTAAGAGTTTGAAACAGatggttgccatgaggacaatgctgaatccatgagggtgcaaaatgtttgctgtgtttccagcatcttcttgtcctcacctctcaggctggttttgggacttcttcagttagaatctggaggttagaattctgcacgtgcttggggaacccaatgtaaggaacaaatggcaaaaccccctcttggggcttagaatgctctggccattgtgacatgagcttccatcccatgatagtggtgggctttctccacctctctgcccaggcaacagctgagaagatcagatacagtgggtgggggagctctacagacgtcacaagcttgtgaggccacagacagcttgtgaggcaaccccagtacattcagctcattggagagaatggccaaagggaggcgtttcaacccgtctttgaataatgacggaagatgggtaaggaaggatgcattcctgaaagcttcctcatgaaatgtggctgttgttgagggcacagatctgggctggattcccttctgctgcctctggtagcttgacctttgaaaagactctgggccttggtcttttcacctgtcaagAGGGAAGCTCAATATTCTTTTGAGGATTCAGTGAACACACATGAATCTGAGGGAGGCAGTCCAAGCCTGTAATCTAAGTACTTGAAAGGCTGAGGCAGGAGGATT
A 571 bp deletion beginning at Chromosome 7 position 133,601,825 bp and ending after 133,602,395 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44361 | GGT TAG CTC CAG AGC AGT ACT CA | Common | A | |||
44362 | AAA CAT TTT GCA CCC TCA TGG | Wild type Reverse | A | |||
44363 | CCC TCA GAT TCA TGT GTG TTC A | Mutant Reverse | A | |||
44364 | Fluorophore-1 | TGG TTG CCA TGA GGA CAA T | Quencher-1 | WT Probe | ||
44365 | Fluorophore-2 | GAA ACA GAG GGA AGC TCA ATA TTC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44361 | 0.40 uM |
44362 | 0.40 uM |
44363 | 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 |