Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination. The transgene genotype is determined by comparing ΔCt values of each unknown sample against known homozygous and hemizygous controls, using appropriate endogenous references.
>chr13:30935304-30935453 150bp TTATGTTCTTGAAGTGGAGGGATTC TTATCTGTGAAACCACTGACTGAAGC
Mutant= 156 bp
Wild Type = 150 bp
Wt Sequence: ttatgttcttgaagtggagggattcatatttagggatatcccaCAtgggtcatttactgtattatttattttgattattaactttttatttttatggtaatatgtatgaggcttaacaagttttgcttcagtcagtggtttcacagataa
Mutant Sequence: gtgaaatcctaaaccaaaaagtgttacttttgccattatagattctcagTAtgggtcatttactgtattatttattttgattattaactttttatttttatggtaatatgtatgaggcttaacaagttttgcttcagtcagtggtttcacagataa
346 bp deletion beginning at Chromosome 13 negative strand position 30,935,755 bp ATTTGTTATAAGGCCAGCAG, and ending after CATATTTAGGGATATCCCAC at 30,935,410 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
35761 | TTA TGT TCT TGA AGT GGA GGG ATT C | Wild type Forward | A | |||
35762 | TTA TCT GTG AAA CCA CTG ACT GAA GC | Common | A | |||
35763 | GTG AAA TCC TAA ACC AAA AAG TGT | Mutant Forward | A | |||
35764 | Fluorophore-1 | AGG GAT ATC CCA CAT GGG TC | Quencher-1 | WT Probe | ||
35765 | Fluorophore-2 | TGC CAT TAT AGA TTC TCA GTA TGG G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
35761 | 0.40 uM |
35762 | 0.40 uM |
35763 | 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 |