Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr15:76292776+76292870 95bp AGGAACAGGGAAGGGATGAT CTCTACTGTCCTGCCCCAAG
Mutant= 102 bp
Wild Type = 95 bp
Partial Wt Sequence (deletions in lower case:
GCCCGGCTCACTCCTCCCAGCCAAAAGTAGAGAGCCAGGTTAGCTGTCTTTGAGTACGACCACTAGGAGccgctcttctggtgtggggttggcggcagagagggacagaccaacatccagaaccctctaaagtggcacagaatgctctctgtgctgaatgcctctggctactccacagatgccctcatccaacccacctaaggaatgatgctcagagagggcaggtcaagccagccagctcagcagacagataccttgaccagacatgagtgggtctttgtgttctttctgtatccacaggtgtattcctgcccccagcttcagcagcagcaaaggagccctgctcagaagacttggggatggtagcactggcacctctggctgacatgttgaataccccacagctcagtcctgcagcaggctcccttgtgaaccccctggctgctactcttaacccactgctgtctggccaaatacccttgttgcagaacaaccagtttgccaatcttgtgccatgctccatgagcaaccaactgaccaatcctactacagtttccccaggagtcaccttggccagttctctgggtttgccctctactggacccctgaacagtcaaatgactagtcccatgactgtacccccagggaccactctggccagctcactgggcctgacctcaactggttccctaacaacaagcagccggctggtggg.........................ttacttggtaatgaaaacagaagttctgggagcggaggaaaaaaaagagggagtaggatcaggaacagggaagggatgatgcagaaagaaaggatacttccctgttcttagCAAAGGGGTAGGGAGCCTCAGGGCCTTGGGGCAGGACAGTAGA.
This is a 9510 bp deletion beginning at Chromosome 15 position 76,283,317 bp and ending after 76,292,826 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46672 | Fluorophore-1 | CGA CCA CTA GGA GCA AAG GG | Quencher-1 | MUT Probe | ||
48499 | TCC TCC CAG CCA AAA GTA GA | Mutant Forward | A | |||
48500 | CTC TAC TGT CCT GCC CCA AG | Common | A | |||
48501 | AGG AAC AGG GAA GGG ATG AT | Wild type Forward | A | |||
48502 | Fluorophore-2 | AGA AAG GAT ACT TCC CTG TTC TTA GC | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48499 | 0.40 uM |
48500 | 0.40 uM |
48501 | 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 |