Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr11:85216246-85216393 148bp TTCCTTTCTTGTCTCATGTGGA AGGTTTTTGTTGTAGTGGTGGTG
Mut= 148 bp
Wt= 148 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GGCTCGGCGTCCGGGGGGCCTGAGGCCCGGAGTGGGGCCGGGCAGAGGGGCGCAGCGTTGCCGATTCGGGGAACCGGGCTAATAATTCCTTTATTAGGATCCATTTTTTTCTATCACAAAGTTGAAAGTTAAAACGTTTGAAATAGTTATTTTTCTTTTTTTTATTATTATTACAGCTTTACCAACAGGGAcgcttgtgtcagctcggcagtgaattctgtgaattggaagtttttgctaaagttttgagagctttggataaaaggtaaatttgtgtgtatgtgtataagctttcttgcttccttccttcctttcttgtctcatgtggatcaagatggccttgaacttgttaaacttctgatcctcctgcctctatttaaatgcttggattacagataatggacagggtcacccccttgcccccaaaaTATCCACCACCACTACAACAAAAACCTGCTAAAATGTATATAACATAAAATTTGCCACTTGTATCATTTTAAAATGCATAATTCAAGGACTAGAGAGATGTCTCAGTGGTCAGGAGCACTGGCTGTTCTTCCTGCAGACCTGGGTTCAATC
This mutation is a 237 bp deletion beginning at Chromosome 11 position 85,216,273 bp and ending after 85,216,509 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
41163 | TTC CTT TCT TGT CTC ATG TGG A | Wild type Forward | A | |||
41164 | AGG TTT TTG TTG TAG TGG TGG TG | Common | A | |||
41165 | GAA CCG GGC TAA TAA TTC CT | Mutant Forward | A | |||
41166 | Fluorophore-1 | CAA GAT GGC CTT GAA CTT GTT AA | Quencher-1 | WT Probe | ||
41167 | Fluorophore-2 | TTA CAG CTT TAC CAA CAG GGA TAT C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
41163 | 0.40 uM |
41164 | 0.40 uM |
41165 | 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 |