Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
chr13:34128761-34128880 120bp TCGTGTTCGGTGAGTATGTG CTTAGGCGCCACTGCTATGT
Mut= 118 bp
Wt= 120 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
AGCAGCTGTTCTCATGGGAAACCGCCCAGTTTCAACTTTCTGATCCTCTACAATGCAAGCCCTAAGGCTGTGCCTGGGTCATGCCACTCATTAAAGCTTCGGCAACGTCCTGTTGTCAAAACTGTGGGTGATGGGGGGGCTGCTCATTGAGAttgggggtagggttgtgcatagctaaatacagtctttttatgtcacagttttgggaggtcatcagtgatgagcatggtatagaccccactggaagttaccatggagacagtgatttgcaactggaaagaatcaatgtatactacaatgaagcaactggtaattacttttaacaccccatcttcaattctggcatccctaagctctgctcttatttgaacccaggagaaggtggcttattttaggatgaccatttttggggtggggtagcctcttctgtgccaaccaaaccctttgaaagcttggtcataatagacctttctttgtttgggggtaccaccaaacaggaaggaggtgggaccgagccctactctatagaagatactgcctggagggctaagccagctcttgttcttctgcaggtaataaatatgtgcctagggccatcctggtggacctggagccaggcacaatggactcagtcaggtctggaccatttgggcagatcttcaggccggacaacttcgtgttcggtgagtatgtggcatagctgaagacagaagagcacaaggtgttaattatcactagcacaaagggTGGAGGAGTGTGATAGACAGGGAGCATACATAGCAGTGGCGCCTAAGCCTTATCAGGCTTTGAGGCAATTGCATCTTAGCTTTTTCAAAGGCAGGTGTGCTCTGTTAACTCATTTATAATTGTATTGATAACCAAATAGTTTATTGCTTTATT
This mutation is a 595 bp deletion beginning at Chromosome 13 position 34,128,808 bp and ending after 34,129,402 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
42858 | TCG TGT TCG GTG AGT ATG TG | Wild type Forward | A | |||
42859 | CTT AGG CGC CAC TGC TAT GT | Common | A | |||
42860 | TGC CAC TCA TTA AAG CTT CG | Mutant Forward | A | |||
42861 | Fluorophore-1 | CTG AAG ACA GAA GAG CAC AAG GT | Quencher-1 | WT Probe | ||
42862 | Fluorophore-2 | CTG CTC ATT GAG ATG GAG GAG T | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
42858 | 0.40 uM |
42859 | 0.40 uM |
42860 | 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 |