Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr18:89559872-89559989 118bp GACCCATCTACAGCTTGAGGA AAGCTTTCTACAACACATTCTCATT
Mutant= 108 bp
Wild Type = 118 bp
Wt Sequence (deletions in lower case):
CACACATACTATCCATTAAAATAAATAAAAAATTCTTTCTTGTTGGAGATGTAGTTCAGTGATAGAATGttttattagcacaaacaagactctaggggcttatccccagaacttcaaaagtaaaaagaaaaaaatatttttctcttttaaggtaactgaactgcacaacatcaaaaatataaccagactgcctcgagagacaaagaagcatgcagtggcaattatctttcatgacgaaacgtcaaagacatttgcttgtgagtcaggtaagcaatttcagacccatctacagcttgaggaaattgagttgcttcagagacaatgactcacacagatgaagacaaaggaagtctgaatagggtgGCATATTGTAATGAGAATGTGTTGTAGAAAGCTTTGTAAGAAGTTCAACATACTGATATGCTGTAGAAACAAATGTTTTTGCATTTC
This is a 294 bp deletion beginning at Chromosome 18 position 89,559,906 bp and ending after 89,560,199 bp (GRCm38/mm10). There is a 5 bp insertion (GGTAG) 106 bp before the deletion.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46702 | GAC CCA TCT ACA GCT TGA GGA | Wild type Forward | A | |||
46703 | AAG CTT TCT ACA ACA CAT TCT CAT T | Common | A | |||
46704 | TGT AGC ACA CAT ACT ATC CAT TAA AA | Mutant Forward | A | |||
46705 | Fluorophore-1 | GAC AAT GAC TCA CAC AGA TGA AGA C | Quencher-1 | WT Probe | ||
46706 | Fluorophore-2 | TGT AGT TCA GTG ATA GAA TGG CAT ATT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
46702 | 0.40 uM |
46703 | 0.40 uM |
46704 | 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 |