Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr2:25223685-25223780 96bp GTCCAACTGACTGCGGGTAG CCTGCCAATGTTCAAGGAGA
Mut= 97 bp
Wt= 96 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GTGCGCCTGCTCCTCCCGAAGTGCTCCTCTTCTACAGCTGTTCCGCAGTCGCCGCCGCCATGAGGGAGATCGTGCACCTGCAGGCTGGGCAGTGCGGCAACCAGATTGGCGCCAAGGTAGGAGGCTAGGTTTCTGCGAGCCAGACGCCACTGGGACAGCCAGCCGGGGAAGATGGCGGAAGcggcccggagcgtcgcccttcattgcgtcctggataccggcgtacgctgggccgagacgtagcgccggtggctggtgtcggtggggcggacggtgcgcggcccttagggattcgtcgccctggcgctcctgagactggagggagggggagggcctgccgcacccatccggggcggggctgccttggagcgcaccggccggccggcgtgactcagctaacgcccgactcccttgcagttctgggaggtaatcagcgacgagcatggcattgatcccactggcacttaccacggagatagcgacctccagctggagcgcatcaacgtgtactacaacgaagccaccggtaaggctcctctaccccatccatcctcctcaccacccttccagtttgagcacacttgacgtccccctgtcccaaccaggtggcaagtatgtgccccgcgccgtgctcgtggacttggagcccggcaccatggactcggtgcgttcagggcccttcgggcagatcttcagacctgataacttcgtctttggtgagtgtggtggaggtgtggacgggcttgctacccatgaggctcaaaggtcaaggggctgacctaaagttcttccttaatagccaccaggtgtacactttcttgccctctgagtcactcaatcccttggccgaaagcagctttaggaggaaggtccaactgactgcgggtagattagttgctagtagagagtatgagaagacctgaCCTAGTGGCAGGGGCCCTGTACTCTCCTTGAACATTGGCAGGGGTGCCTTTGGAAGTATTTACTGTCTTAGCTCTAGTGTGTTCTGTACATCTCCGTCTGGTCTCCCCCACCCCCCATCCCCAATCTATGTATCCCTGCCTGGCCTCTTTAACTGTAGACCAGGCTGACCTTAAACTCA
This mutation is a 742 bp deletion beginning at Chromosome 2 position 25,223,727 bp and ending after 25,224,468 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
38908 | GTC CAA CTG ACT GCG GGT AG | Wild type Forward | A | |||
38909 | CCT GCC AAT GTT CAA GGA GA | Common | A | |||
38910 | AGG TTT CTG CGA GCC AGA C | Mutant Forward | A | |||
40007 | Fluorophore-1 | TGC TAG TAG AGA GTA TGA GAA GAC CTG | Quencher-1 | WT Probe | ||
40008 | Fluorophore-2 | ATG GCG GAA GGC CCT AGT G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
38908 | 0.40 uM |
38909 | 0.40 uM |
38910 | 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 |