Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr18:43581792-43581904 113bp AGTGAGCATCAGTCCCACCA GAAAATACCCAACCGGAGTC
Mut = 117 bp
Wt = 113 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
TCACCTCGCTGCCTCTAGGTATCAAAGCTTGAAAGAGAGAAGACTCAAGAAGCAAAGAGGATTCGTgagttggagcagcgcaaacacacggtcctggtgaccgagcttaaagccaagctccacgaggagaagatgaaggagctgcaggccgtgagggagaatctcatcaaacagcatgagcaggaaatgtcaaggacagtgaaggtgcgggatggggagattcagagactcaagtccgcgctgtgtgctctccgggatggcagcagcgacaaagtaaggacagcactcaccattgaggcccgggaggaggcccggaaacagttcgatgcagagcgcctgaagctcctgcaggaaattacagacttgaaaactgccaagaagcaggtggatgaggctctaagcaatatgatccaagcggataaaatcaaagctggggaccttaggagtgagcatcagtcccaccaggaagccatctcgaagatcaagtgggaatccgaacgggatatacggAGGCTGGTTAGTACTAATAAACCGGGGGACTCCGGTTGGGTATTTTCTTGTCGGTTACCATAAACA
This mutation is a 444 bp deletion beginning at Chromosome 18 position 43,581,839 bp and ending after 43,582,282 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
51015 | GAA AAT ACC CAA CCG GAG TC | Common | A | |||
51016 | AGT GAG CAT CAG TCC CAC CA | Wild type Forward | A | |||
51017 | CTT TCA CCT CGC TGC CTC T | Mutant Forward | A | |||
51018 | Fluorophore-1 | CCA TCT CGA AGA TCA AGT GGG | Quencher-1 | WT Probe | ||
51019 | Fluorophore-2 | AAG CAA AGA GGA TTC GTT AGG CT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
51015 | 0.40 uM |
51016 | 0.40 uM |
51017 | 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 |