Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr9:110312428+110312539 112bp AGAGAGAGGGTGAGGAGGTG CAAATCCTCTTCCTACAACCAGA
Mutant= 93 bp
Wild Type = 112 bp
Wt Sequence (deletions in lower case):
GGTTGCGTTTCCAGTATCATAGCCAGGGCTACATAGAGAAATCCCATCTCAAAAAACCAAAATAACAAAACAAAGCAAAACAAAAAATAGAGTTCAAAGTTGTCCTGGGTTATACAGGGAGTTGAAGACCAGCATGTGCTACCCGAGACACTGTGTCATAGAGAGAGGGTGAGGAGGTGCGCCAGAGTGCAAGGCctcggctaggccttcctcatagctcctgtctccagaggattgaaatagttatgtctggttgtaggaagaggatttgtttaaaaagtcatttattttgttttttagctaattgtaaagtctgctttgtggcacttgtccagtccttcagccattacaatattgtgggacagaaactggtaagtgtttcagccctctgtggtgacatcccctcaaaggagcatgtgtcacttgtggctaggactgggactttcttcttcttcttcttcttctccttctccttcttccttcttccttcttccttcttcttccttcttcttccttcttcttccttcttcttccttcttcttccttcttcttccttcttcttccttcttcttccttcttcttccttcttcttcttcttcttcttcttcttcttcttcttcttcttcttcttcttcttcttcttccttcttccttcttccttcttccttcttccttcttccttcttcttctttttttatttattattttttttattttttgagacagggtttctctgtgcagtcctgtcctggaactcactctgtagacccaagtaaacaaacaaacaaataaaacaaccaaacacaagctgggggtatgacttagtggtaggactgggtttaatccccagttctagaaaacaaaaggggaagaggacattttggcttggtatgatggttccaaggcctcaaactcagaaattcaccggcctctgcctcccaagtgctgggattacaggcatgtgccaccactgcctggctgaagctgggcttttctgcttaggttgtggagaacctaacaggaaggactgaggatattttgggactctgaaccccttatccaaaggatgggcgtgttttattgaaagcatatagaaccagacctagtatatgtagttgtcagtgactcaaacccttctcctgattgcggccagttaagattttcttgcctagctgagtatgttttcacatgactttaatcccagcactaggaggtagaggcaggacacaagatcggggctgggactgcagaggtggctccgtggttagcactgctgcagaggacctgagttaggtgcccagcacccccgtcaggcagcttacagcctcctgaggcagctgcacttgcatgcagagacatttacacataattaaaaagtaaacataacctcagcattcaggaagcagagacaggccggcctggtcacagatctagttccgggacagctagggatacacagacactctgtcttggggggaaaaaaaaggtcacagtcatgttctactgcattttgggtcaaagcttgcttgggaggatacccaagtccttgttgcaaatatcagatagcagagtggaaatagacaggaacacttaagcggggtttggagtatgtcttgtaccctcttattagctagtatgtagtttctctgaaaggctctccattccccagagggcacagcaattcactctgctctttgtcttgtctagggtgtcagcttgacagcggctcgggacagagggcagcttgtgttcctggagggtctcaagtcctctgtggaggtcctcttccactctcaggacgagcctcaccccctgcagtttctcaggtcagaaccatagtgctggggtggggcaggcaagggctgctgtgttgtgtgggaaggcagaagtgacttcagggatcagactctttccccgtgttcctgtgtgtcagatagcccctggctgccatcatgaagctgacggtgggtggtggtccccagagctgtgggaagcgtccgtcatttgcTGGGAGGATGGCGCGGTGATGTTACCCTGCTGTTTCGCTGATGGGGGCTTAGACCTCTCATGCACCTACTTTTGCTCTTTCTTGCCTTCATGTAGAAACTTCATCTTATGGGCCAGTGACCTTATTGGGACATAGCCATATTTGTTTGTCATTGATTGTCCCTGGAATATTGGGGGGGAAAATG
A 1813 bp deletion beginning at Chromosome 9 position 110,312,464 bp and ending after 110,314,276 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
49602 | AGA GAG AGG GTG AGG AGG TG | Common | A | |||
49603 | CAA ATC CTC TTC CTA CAA CCA GA | Wild type Reverse | A | |||
49604 | GAG GTC TAA GCC CCC ATC AG | Mutant Reverse | A | |||
49605 | Fluorophore-1 | CGG CTA GGC CTT CCT CAT AG | Quencher-1 | WT Probe | ||
49606 | Fluorophore-2 | TGC AAG GCT GGG AGG AT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
49602 | 0.40 uM |
49603 | 0.40 uM |
49604 | 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 |