Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr7:5070062+5070154 93bp AAGCCTGGGCTGTCATTG AACTCTGACAATGTTGGAAAGG
Mut = 93 bp
Wt = 93 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
ATTGCCCATAAGAGTCCACTTTGATTTGTACTCTTGGAGACTAATGTCACTTCATCAGAAAGCCTGGGCTGTCATTGCCACgtaacttagttgacctaacttttctatgctggcttctcagcagttggtccctttccaacattgtcagagtttatgatagtgtgcttgctatctgctgggtgcgaggatattgctttctagagtgtgagcttctagaagctatctggtagtgcttgttcccattgttcattatgggtttggtactatgtttgtttatataagtgggaggaaagttaagagtcttgggggctggtctagtcccaacctaccccttagctctgttgtttctcttccttcagttccggtcagtggatgagacgacccaggccatggcatttgatggcatcatcttccagggccagtcattgaagattcgaaggcctcatgactatcagccattgcctggcatgtcagagaacccctctgtttatgtgcctggtgagtgggaattcgttgggtgggtggtatggggagtcactgtttcctcaacccaggttcctgtacttaGTCCAGCTAGAGTTGCTTTGGAGATAGTGTGTGTGCGCTGTGGATGTTCTTCTAGCTTGGCAGGTGTCCTGGGCCTTTGAGGTGGTGATCTGTGATCTTCAATCTTAAAGTTTAGCTTAGCTTGTAAATGTCAGAGCCAGGTCCAGATCAGAGGTGTCCTGGAAAGTGTGGGACTTGCAGTCTGGGCATTGTTTTTACCTTGTCCCCTTAGCCCTGGGATTTTAGGGCTTGATGTGTTGT
This mutation is a 486 bp deletion beginning at Chromosome 7 position 5,070,084 bp and ending after 5,070,569 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
50900 | AAG CCT GGG CTG TCA TTG | Common | A | |||
50901 | AAC TCT GAC AAT GTT GGA AAG G | Wild type Reverse | A | |||
50902 | CAG GAC ACC TGC CAA GCT A | Mutant Reverse | A | |||
50903 | Fluorophore-1 | CTA TGC TGG CTT CTC AGC AGT T | Quencher-1 | WT Probe | ||
50904 | Fluorophore-2 | ACG TCC AGC TAG AGT TGC TTT G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
50900 | 0.40 uM |
50901 | 0.40 uM |
50902 | 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 |