Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr18:14679365-14679469 105bp TTTCAGATGCTGGATGAAAAC ACCAAGAGCTTGTCTGTCTTACTGC
Mut = 139 bp
Wt = 105 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
AACTAGATGGTTTTATAATCTGAAGTGGCTTTGAGGAAACAATGTTTCGCTTCTTTGCAGTGTCGTGttcatggataccttctcttggcttcttggttttgaattttactggatatacaggtttataatgaactctctggcttagttttgagttttactcgatgtacaggtttataatgaactctctggttttaatttcagatgctggatgaaaacaaccatcttattcagtgtataatggactatcagaacaaagggaaggcctcggagtgctcgcagtaagacagacaagctcttggtcttgggtcttgTGGTTGTTACTTGTTACTTTGTCTTTGAGTTATTTAT
This mutation is a 244 bp deletion beginning at Chromosome 18 position 14,679,354 bp and ending after 14,679,597 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
50065 | TTT CAG ATG CTG GAT GAA AAC | Wild type Forward | A | |||
50066 | ACC AAG AGC TTG TCT GTC TTA CTG C | Wild type Reverse | A | |||
50067 | AGT GGC TTT GAG GAA ACA ATG | Mutant Forward | A | |||
50068 | GGT GAC AGT ACC ATG ACT CTA CAA G | Mutant Reverse | A | |||
50069 | Fluorophore-1 | AAA GGG AAG GCC TCG GAG T | Quencher-1 | WT Probe | ||
50070 | Fluorophore-2 | TGC AGT GTC GTG TGG TTG TTA CTT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
50065 | 0.40 uM |
50066 | 0.40 uM |
50067 | 0.40 uM |
50068 | 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 |