Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 94 bp
Wild Type = 124 bp
chr7:30161703-30161826 124bp GGTAAAAAGCCTTATCAGTGCAG GAACACTTCCCCGTACTGAAG
Wt Sequence (deletions in lower case)
AGCTGCAAAGCCCTGAGCCGGATGGCGGCGCGCTCGGCACTGCTGGAGGAAAGCAAGTGAACTTCGCAGAGACAACATTGGCTATCAGTTAAACTTGGCCTGGGAAGAACGTTTGTTCTGATAAAGCTCTCTTAAACCTCCTCCCTTGCTCTGCATTTGAGTGAGCAGGCAGGCACAGAAAGAAGCCCTCGGACTGTAAGAAATGTGGAAGCATCCTCAGTCAAGAGCAGAGCCTCGTTTGTCTGGACTTCCCTATTGCAGAGAAGCGCTGTGAATGAGATCACTCTTCCACTCAGATGTCACAGCTCAGCCAGCAGAGAATCttaagcgggggaagcccctttgcctgtaaggtatgtgggaaactcttcagccacaaatcgaatcttacagagcacgaacatttccatagtagagagaaaccgtttgagtgtaacgagtgcgggaaagcctttagccagaaacagtatgtcatcaagcaccagagcactcacagcggagagaagctattcgagtgcagtgactgcggaaaagcctttagccagaaggaaaacttgcttacgcatcagaaaattcacactggagagaaaccttttgagtgcaaagattgtgggaaagccttcattcagaagtcaaacctcatcagacatcagagaactcacacgggggagaagcccttcatatgtaaggagtgtgggaaaaccttcagtggcaaatccaatctgacggagcacgagaagattcacatcggagagaagcccttcaagtgcaacgagtgcggaacggcttttggccagaaaaagtacctgataaagcaccaaaacatccacaccggggagaagccgtacgagtgcaacgagtgcggcaaggccttctcgcagcgcacgtcgctgattgtgcatgtgagaatccattcaggcgataagccttacgagtgcaatgtatgtgggaaagccttctctcagagctcatctctaaccgtgcatgtgaggagccatacgggtgagaaaccctacgggtgcaatgaatgtgggaaggccttttctcagttctcaaccctggccctgcatctgagaatacacacaggtaaaaagccttatcagtgcagtgagtgtgggaaggctttcagccagaagtcccatcacattagacaccagaaaatccatactcattaaacgCCCTGAATATCTTCAGTACGGGGAAGTGTTCATTAGGAATTTATACCTCATAGCGCATCGTTTGGTCACTCTGGCTAAGCACCATAAATCAGCCAAACATTTAACAGAAGATGTCGTGAACACCGGAGAATTCACAATGAAGGGAAAGACATGTGTATGATACAGGCCTGCAATCAGCGAATGCAGCGGTGGAAAAGCAGAGCAGTGCTGAACTGTGAACACAGTCGTCTGCGGTCAAGAGTGGCAGGCAGGCAGGCCTGTTGTGA
This is an 855 bp deletion beginning at Chromosome 7 position 29,861,159 bp and ending after 29,862,013 bp (GRCm39/mm39).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
58397 | Fluorophore-1 | AGC CAG CAG AGA ATC CCC TG | Quencher-1 | MUT Probe | ||
58398 | Fluorophore-2 | AGT GTG GGA AGG CTT TCA GC | Quencher-2 | WT Probe | ||
58399 | GAA CAC TTC CCC GTA CTG AAG | Common | A | |||
58400 | GAG AAG CGC TGT GAA TGA GA | Mutant Forward | A | |||
58401 | GGT AAA AAG CCT TAT CAG TGC AG | Wild type Forward | A |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
58399 | 0.40 uM |
58400 | 0.40 uM |
58401 | 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 |