Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr6:34905318-34905417 100bp TCTCTCCTGGATGGGGAACT CTGTGGGGACCAGTCCTTCT
Mut = 105 bp
Wt = 100 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
ATTATTAGCTGTCGCTCTTTCATTTGCCCACCCTTTGATGAGCAGAAATGGTTTGGATGTGGCGTGTCTAGGAAGCTCCCTCaacagagggaaggtcctgtgatgtcactggctcactgggctttgcccctccatagctttttgcactgcaagctgaaatccatcgactaaagaaggaagagcagcagcaggaagaagaagcagcagctttggtccagcataagctgccgccttatgtttccagtatggaccgccttggggactcggagctgtgagtaggcttggggttcgagaggggtgggggctgccatatgtgtgaggtcccaattttttaaggttctctcctggatggggaactgtatagctagctgtgtcaggtatcatgataacggagtccagggaagctgcttcATAGGTGAGAAGGACTGGTCCCCACAGGTGGACATGTTCCTCAGGAGAAGCTGCTGGGGTTATAAAGAGGTAGACAGTATGGGAGAGAAGTTT
This mutation is a 329 bp deletion beginning at Chromosome 6 position 34,905,345 bp and ending after 34,905,673 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
52299 | CTG TGG GGA CCA GTC CTT CT | Common | A | |||
52300 | TCT CTC CTG GAT GGG GAA CT | Wild type Forward | A | |||
52301 | TTA GCT GTC GCT CTT TCA TTT G | Mutant Forward | A | |||
52302 | Fluorophore-1 | ATG ATA ACG GAG TCC AGG GAA G | Quencher-1 | WT Probe | ||
52303 | Fluorophore-2 | CGT GTC TAG GAA GCT CCC TCA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
52299 | 0.40 uM |
52300 | 0.40 uM |
52301 | 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 |