Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 106 bp
Wild Type = 125 bp
Fam=Mut
Hex=Wt
X-linked
>chrX:36479941-36480065 125bp CCTTCTGAGCTCTCTATGGTATCT GACTTGACTCCCATGCCACT
Wt Sequence (deletions in lower case):
TGTGTATGTATGAGTGTGTGTGTAAGAGAGATGTGTATACGTTGACTGGGTCCACACTCTTTTTTTTATTCTCTCTCTCCATCTCAGAGATGGCTTTCAGTGTCAGCCCATCTGCATCTCTGCATCCTTCTGAGCTCTCTATGGTATCTATGTTATGAAATACCTCACCCTTCAattcttaatccctcgcactgcttttacaagacttttcctttccacataggccaaagagtggcatgggagtcaagtccatatcccatgacagtgtcttctgtttggatcctgagcctgaaaaaggagcaggcaagctacactcttctccagctccccacagaagcaaatcactaaaggtaacaatgagtctgacctcactctgattagacactgttctatcctgatcccttctCAAAGGAGGGAGGAATTTGATCCTGATTATCAACTAATTCTCTCTCACAAGACTATCTTAAATTTCAAATAATACAATATATAAATAGGCATTTAACTATGTGACATGATGAACACATAGTGTTGGCTGTTGTGTGCTTTTATCTATCACACCCAAAGATACCATAATTATTTTGATAATGGACAGTTTACTCTTTTTTGTTGTTGTTGTTGGTTCTTTTTTGGAAAGTTTACT
This mutation is a 232 bp deletion beginning at Chromosome X position 36,479,785 bp and ending after 36,480,016 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
62635 | CCT TCT GAG CTC TCT ATG GTA TCT | Common | A | |||
62636 | GAC TTG ACT CCC ATG CCA CT | Wild type Reverse | A | |||
62637 | GAT AGT CTT GTG AGA GAG AAT TAG TTG | Mutant Reverse | A | |||
62638 | Fluorophore-1 | TTC CTT TCC ACA TAG GCC AAA G | Quencher-1 | WT Probe | ||
62639 | Fluorophore-2 | TCA CCC TTC ACA AAG GAG GGA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
62635 | 0.40 uM |
62636 | 0.40 uM |
62637 | 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 |