Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr17:32335340-32335455 116bp CCTTCCTTATGGGACTGGA TGCTAGACTGGAGGCCACAC
Mut= 114 bp
Wt= 116 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GGGCCAGTGCATGCCTGGTGCCTCCCGGCTGCCCTCCCTCTTCTCCCAGAACATCATACCCGAGTATGGCATGTTCCAGGGCATGCGTGGTGGGGGTGCCTTCTCTGGTGGCTCCCGATTTGGCTTTGGATTTGGCAatggcatgaagcagatgaggcgtacctggaagacctggaccacagccgacttccgggtgaataagctgacctacccctcctgatccatgctggaatatggctacaatctctgagttcactcctcctttcacttccagaccaaaaagaagaagagaaagcagggtggcagtcctgatgagccagacagcaaagctaccagaacagactgctcagacaacagtgactcagacaatggtaagttggggaatgtggtgtcctgctccctgtccccgtggggcccctggattgaacttgactgtcctctccttgcagatgaaggcactgagggggaagctgcagagggtactgaaagtgctgaggctatggagaaaggctcccgagcagtaagtaaccctagacctcaggctcttgctctctgcaggtggggcataggactccagagctggcttaaagacacacagccttgctacaagggtcacaggcaatgtggtcttacctgtatgcatggctagtgagtggccagcacaaggaggctgggtatctagggaaaagtacagaccacttcactaacagtgtgtttccttgggagaaatggggcgatgcaggcctctgtgcagcaggtagtgagtgggaaggtctgacgctcccttaatcctttagagcaggtataagtagctgtgccctttttaaaattgaattctaaaaattgtatattaggaaggagaagacgaggagggaaaagaggatgggagagaaggcaaagaagattcggagaaaggtaagtcttccatggttacctaggtactgggaaagctcttccaggtgcttgtagtgggaggaaaccagctgagggcagggttgtagcggaaagatggctggctgggaccttccttatgggactggatatatgttagcaaggcctcttcttactctgtgccctcccagcctgcctccgtttttcctaGGCAGAGGTTATGGATGGTGTGGCCTCCAGTCTAGCACTTTGGTTTTGCCTTCAGGTGGGGGCAGCAAGCAAAAGTGTGGGGAGCCCAGGCACCCCCCCCCTCAGTTCCTCAGTAATAGACAGTTCCAAGTCCTC
This mutation is a 964 bp deletion beginning at Chromosome 17 position 32,335,377 bp and ending after 32,336,340 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
43614 | CCT TCC TTA TGG GAC TGG A | Wild type Forward | A | |||
43615 | TGC TAG ACT GGA GGC CAC AC | Common | A | |||
43616 | CGA GTA TGG CAT GTT CCA G | Mutant Forward | A | |||
43617 | Fluorophore-1 | CTC TGT GCC CTC CCA GC | Quencher-1 | WT Probe | ||
43618 | Fluorophore-2 | CTT TGG ATT TGG CAG GCA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
43614 | 0.40 uM |
43615 | 0.40 uM |
43616 | 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 |