Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 110 bp
Wild Type = 137 bp
>chr1:172491303+172491439 137bp CCACCAGGAAGTCTCACCTC GCCACTCGCTTTTCCAAGT
Sequence (deletion in lower case):
AGAACCGATTAATTGATGGCCCACTGCAGTATCGTTCTCCTTGGTACTTGAGAAGTACAGCCCTGGGTAATACAGTTAAAGCCAGAGACCCTGACATGAGGAAGGCCCACCAGGAAGTCTCACCTCCCTTTCTGACCTCCCTGgaccctcccgcacatacagtttctcagcagaaggaatcagggtgggtaggcttgcctgccatgcctcgtgaaacagggctgacttggaaaagcgagtggcctctggaacaggatgggcttttatcacacatcccaattcgggtccttaaggtcctgtagcaacttggcccatgactacaaagggcagaaccaggaagaggtgtagaaaggccctgagggttctgggaattccggaatgggctgaggacacctttctgccttggtacttctcccaggaccccctgtcattgtggtgccccccagcaacagtacagtcaactcctctcaggatgtttccttggcctgccgggctgaggcataccctgctaacctcacctacagctggttccaggatggtgtcaatgtcttccatatcaggtagggcaacctatctcttgggtagtggaggcaggggattgatgtgtagtctctgggagagctccttgtctaaggactaggttcctcctcacacataccatattgcagccgcttacagtctcgagtgcgcatcctggtagacgggagcctgtggctacaagccactcagcctgatgacgccggccactatacctgtgttcccagcaatggctttctgcatccaccgtcagcttctgcctatctcactgtgctctgtaagtccccatgctccttccacagcctgctcctccgcactgagacagactgatgaccttgtcctccaacttgtctctgcattcctccagacccagcccaggtgacagtcatgcctcccgagacacccctgcccactggcatgcgtggggtgatccggtgtccggttcgtgctaatcccccactactgtttgtcacctggaccaaagacggacaggccttgcagctggacaaggtacaggcttgtgacaggggatgaaggtcttggctaggtgttgtttttgtcagagggccagctgcagctcagcgggaggctgagtttaattcagcgctgtggttgggaaacatgtttcttaccagatgccagtgttaggcagtgactgtggaggtgagaagaagtatccatttccccagaagaagtatccatccttggagtgttgccttgaggcagagctgccacagagactctgctgtcaggccagggaaggaacaagGCTTTTGTTTTTAGGTCCGGTTGGGGATAGACCAGTAACAGATCCTGGGCCTTTGGTGAGGTGGTAGGTCTTGAAGAGTGAGCTGTCATTCCATGCAGAAATCCCTTTGCAGAGGCCGTATAGTGTGCTGACAAAGTGATGAATCGGGCAGTGAGGGGGAGCC
1163 bp deletion beginning at Chromosome 1 positive strand position 172,491,340 bp and ending after 172,492,502 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
37683 | CCA CCA GGA AGT CTC ACC TC | Common | A | |||
37684 | CAA GAC CTA CCA CCT CAC CA | Mutant Reverse | A | |||
37685 | GCC ACT CGC TTT TCC AAG T | Wild type Reverse | A | |||
37686 | Fluorophore-1 | ACC AGT AAC AGA TCC TGG GC | Quencher-1 | MUT Probe | ||
37687 | Fluorophore-2 | CTC CCG CAC ATA CAG TTT CTC | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
37683 | 0.40 uM |
37684 | 0.40 uM |
37685 | 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 |