Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr12:55795118-55795263 146bp CTCCAATTCTCAGACCTAGAACCT CAACCACTGGCTATTGTTTTCT
Mut= 125 bp
Wt= 146 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletions in lower case):
AGTGTAAATTAAGTTTTCTTTTTATCCTGGGGATTGAATCCAAGGCCTAGTACATGCTAAGTGTGGGTTTTCTCATTAAGCTACTCCAATTCTCAGACCTAGAACCTGAATTTAGAATAATTATGCTAAGGAGTAGtgagAAGGTTTATAGTATTgtctatataataaatagaccatgatttagcatttgtctaaaagaatcattgaagaaaacaatagccagtggttgaaatatttaagttcattaagtagttgttaatagtgagtgtatttaaagtatttttctgttccaaatatttcaggtcacaagtctcaaagagaggaattagatgctatactttttatttttgaggtaagtgctatgaatcatactgtttaaactttttcaaaaaattattctgagaatataaagaaatatattaaaattattagacatatagtgaaacatagttaacctgccagccttttGAAGAACTTAATTAACTTTAAAAATGTATCTGGCTGAACTTGTGTATTTTTGAAGTATAATGTATTTTTTAGCAACAAAATTATGCATCTGACAATATAAAATTCATTTGATAGCATTTTTATAAAGTGAAAAAAAATTTTTTAA
This mutation is a 313 bp deletion beginning at Chromosome 12 position 55,794,879 bp and ending after 55,795,191 bp (GRCm38/mm10). In addition there is a 4 bp deletion (TGAG) 15 bp before the 313 bp deletion that will not alter the results of the mutation.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
39256 | CTC CAA TTC TCA GAC CTA GAA CCT | Common | A | |||
39257 | CAA CCA CTG GCT ATT GTT TTC T | Wild type Reverse | A | |||
39258 | TCA AAA ATA CAC AAG TTC AGC CAG A | Mutant Reverse | A | |||
39259 | Fluorophore-1 | AGA CCA TGA TTT AGC ATT TGT CTA AAA | Quencher-1 | WT Probe | ||
41194 | Fluorophore-2 | AGG AGT AGA AGG TTT ATA GTA TTG AAG AAC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
39256 | 0.40 uM |
39257 | 0.40 uM |
39258 | 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 |