Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 134 bp
Wild Type = 134 bp
chr11:58932161-58932294 134bp TCCAGCCTCTCTGTTTCCATA CCCATCCTGGAATATCTTAGTGA
The common primer (primer 60064) anneals over the nucleotide sequence containing mouse genomic variations rs241612121 and rs266215563.
Wt Sequence (deletions in lower case):GCCAGTTTTATCCTGCTTCAGGGCTGAAGGTATAGCGATAAGTCACCCTGGAAAAGCCTTTCCTGACAGACCACCTCCCAAATTATCTGTGGGAGATGGGCAATTATTCATGATGTTGGGGCAGTACCCTgcaggatgagtaatcttataggacacaggg[....]gtcaggggccaaagacatagatgtatttaggtttcgggtaacaatgttctgtccccagaaggccccagccttggatgtagtgaccggcctcaacttgactcgcaggtgggctgggttttcagtgctgaataaagtggtttgctccagcctctctgtttccatacctcctagtctttgtctacacagctgcttcccccttccctgaccagGTTCCAAGGACTTTGATACCTTTGAAAGCAAGATAAGGAGACTGTCACTAAGATATTCCAGGATGGGGTGTGGGTGTGGGTGTGTGTCATCCCAAACCCAGCGTTGGCTGT
This is a 6959 bp deletion beginning at Chromosome 11 position 58,823,054 bp and ending after 58,830,012 bp (GRCm39/mm39).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
60063 | TCC AGC CTC TCT GTT TCC ATA | Wild type Forward | A | |||
60064 | CCC ATC CTG GAA TAT CTT AGT GA | Common | A | |||
60065 | TGA CAG ACC ACC TCC CAA AT | Mutant Forward | A | |||
60067 | Fluorophore-1 | TCT TTG TCT ACA CAG CTG CTT CC | Quencher-1 | WT Probe | ||
61120 | Fluorophore-2 | TTG GGG CAG TAC CCT GTT CC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
60063 | 0.40 uM |
60064 | 0.40 uM |
60065 | 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 |