Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 92 bp
Wild Type = 120 bp
chr18:65625246+65625365 120bp GTCTCAAGTGTTTGGAGTGTAATG CCGAAACACCGAAATGTAAATG
The common primer (*59077) anneals over the nucleotide sequence containing mouse genomic variation rs244015298.
Wt Sequence (deletions in lower case):
GAGAAAGGCGTGGTGATGCAGTGCTCCCACCTGATCCTAAAGCCGGTCCCGGCAGACCAGATGATAGTTCCTCCATCCAGCAATACTGCTGCTTCCACTCTGCAGAGCTCTGTGGGAGCTGCCACACACACTGTCCCAAAAGTCCAGCCTGGCATAGCCGGGGCAGTTATCTCAGCTCCGGCAAGCACACCCATGAGCCCAGCCATGCCCCTAGacgaagacccctccaagctctgtagacacagtctcaagtgtttggagtgtaatgaagtcttccaggatgagccgtccctggccacacatttccagcacgctgcagacACCAGTGGACAAGTAGAGTATCATTTACATTTCGGTGTTTCGGGGACGAGGCTGGAGCCGCGAGTGCTTTGACAAATGTCTTGAATTACGGTGGAGAGGGGTGTGTGTGTGTGTGTTTGTGTGTGTGTGTGTGTTCAGGCAAGCACAGGAAAGGGAAAAGATGGTTTTCATAGTATAGGTGCAGTGCTGAAATACAAGGGTAATTCTTCAGCCAGATAGAAAAGTGTGTGCTCTGGTTGGATTATGTTAGGTAATTGAGTAGGCATTGGAAGTCTGATTCTTAGCAGTAAATTCAGTGTCCTCCCATCATATGGAGCGT
This is a 107 bp deletion beginning at Chromosome 18 position 65,758,287 bp and ending after 65,758,393 bp (GRCm39/mm39).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
59073 | GTC TCA AGT GTT TGG AGT GTA ATG | Wild type Forward | A | |||
59074 | Fluorophore-1 | ACA TTT CCA GCA CGC TGC AG | Quencher-1 | WT Probe | ||
59077 | CCG AAA CAC CGA AAT GTA AAT G | Common | A | |||
59078 | Fluorophore-2 | AGC CAT GCC CCT AGA CCA GTG | Quencher-2 | MUT Probe | ||
59079 | GTT ATC TCA GCT CCG GCA AG | Mutant Forward | A |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
59073 | 0.40 uM |
59077 | 0.40 uM |
59079 | 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 |