Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr4:83482138-83482264 127bp TCATAGAGATGAGCTAGCCTGAG TAAGGAAGCTTAAAGGGCTTGAC
Mut = 139 bp
Wt = 127 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
TGGAGCTGATCTGGGGGCCGGGAGGGAAACGGGCTCTATTACAATAAACTGTAAAATGGGTTGATCCTGTGTTAGACATATTTGGGGCTCTTTACTCTGAGTTCACTCAGGTGTActaaggtttgtaagatctgactttcctttcttacaggtagatgaagttcctgatggagctgtaaaaccacccacaaacaaactacccattttcttttttggaacccatgagacgtaagtcctgctagctttaagctgaaatgacagttaacttgcagaagttaggtataaaacaaaaagcaagtagaaatttcaaaatttaagtgggatgtgacaatgaaattaaaaacttatatttttaaagacatagtctcttgtagccctggttgccctaggatgttccaatgagacccggctagcctgagctcatagagatgagctagcctgagctcatggagatcaacttgttgtttgcctcctATGTTTGGAGATAAAATGCCATACCTGGACTGGAATAGAAAACTTAAAAAGTCAAGCCCTTTAAGCTTCCTTAAAAATTGTAAAATGTACTTTCCCGTTTAATGCCA
This mutation is a 359 bp deletion beginning at Chromosome 4 position 83,482,211 bp and ending after 83,482,569 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
50164 | TAA GGA AGC TTA AAG GGC TTG AC | Common | A | |||
50165 | TCA TAG AGA TGA GCT AGC CTG AG | Wild type Forward | A | |||
50166 | TGT AAA ATG GGT TGA TCC TGT G | Mutant Forward | A | |||
50167 | Fluorophore-1 | ATG GAG ATC AAC TTG TTG TTT GCC | Quencher-1 | WT Probe | ||
50168 | Fluorophore-2 | CTG AGT TCA CTC AGG TGT AAT GTT TGG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
50164 | 0.40 uM |
50165 | 0.40 uM |
50166 | 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 |