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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chrX:147074037+147074167 131bp TGGACTCTTCGAGTTCCCACT CAGGAGGATGGGAGGTGAGT
Mut= 136 bp
Wt= 131 bp
Fam=Mut
Hex=Wt
X-Linked
Wt Sequence:
aggccctgtctatttactgtaggtggactcttcgagttcccacttcccaatgttgagcattttggctaaggtcaccccaattgagtcctaagagtctcttgactcttgggtctctagaattttctagagagcccactcacctcccatcctcctgaggcagtgtagttccattctttctcttggccctctgggcttctctcct
Mutant Sequence:
GAAGGCAGGTCAGAGACCCCACTGGACAAACAGTGGCTGGACTCTGCACCATAACACACAATCAACAGGGGagtgagctccggatccataactTCGTATAGCATACATTATACGAAGTTATTCTTGACTCTTGGGTCTCTAGAATTTTCTAGAGAGCCCACTCACCTCCCATCCTCCTGAGGCAGTGTAGTTCCATTCTTTCTCTTGGCCCTCTGGGCTTCTCTCCTGTCCCCCCATACCTGATCCTGTTTCCCTTTTCCCCTTCCCATCCCCTCTCCTATCCAGGTCCTTCTCTTCCG
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
57761 | TGG ACT CTT CGA GTT CCC ACT | Wild type Forward | A | |||
57762 | CAG GAG GAT GGG AGG TGA GT | Common | A | |||
57763 | CTG CAC CAT AAC ACA CAA TCA | Mutant Forward | A | |||
58328 | Fluorophore-1 | CAT TTT GGC TAA GGT CAC CCC A | Quencher-1 | WT Probe | ||
58329 | Fluorophore-2 | AGT GAG CTC CGG ATC CAT AAC TTC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
57761 | 0.40 uM |
57762 | 0.40 uM |
57763 | 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 |