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.
>chr16:48872792+48872881 90bp GTCCTTTCCCTTACAGCCATC ATCTTGTTGAAAGTCAGCATCC
Mutant= 105 bp
Wild Type = 90 bp
Wt Sequence: gtcctttcccttacagccatccCAtagctcagtgatctcatcttttctgctctttctctccagCCCTAGGATGCTGACTTTCAACAAGAT
Mutant Sequence: gtcctttcccttacagccatccCTgtacataaatcctaaaatcccctgtaccctggtctaagctcttagtcctctccatccctgccttcctgaccagggcttctt
243 bp deletion beginning at Chromosome 16 positive strand position 48,872,815 bp ATAGCTCAGTGATCTCATCT, and ending after GAGCTTTTGCTGATGCTGAC at 48,873,057 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
35773 | GTC CTT TCC CTT ACA GCC ATC | Common | A | |||
35774 | ATC TTG TTG AAA GTC AGC ATC C | Wild type Reverse | A | |||
35775 | AAG AAG CCC TGG TCA GGA AG | Mutant Reverse | A | |||
35776 | Fluorophore-1 | CTG CTC TTT CTC TCC AGC CC | Quencher-1 | WT Probe | ||
35777 | Fluorophore-2 | CCC TGT ACC CTG GTC TAA GCT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
35773 | 0.40 uM |
35774 | 0.40 uM |
35775 | 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 |