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This assay cannot distinguish hemi from hom
Tg= 96 bp
IPC = 74 bp
>chr16:84962983-84963078 96bp CGCCATCATCGGACTCAT TGGATGGATGTGTACTGTTTCTTC
These "transgene" primers amplify mouse. The probe is what makes this assay specific.
Tg Sequence:
Indiana (V617F) 642 of 695aa
Hu WT V TTGTCATAGCGACAGTGATCGTCATCACCTTGGTGATGCTGAAG
Hu V->I TTGTCATAGCGACAGTGGTCATCATCACCTTGGTGATGCTGAAG I716V(FL),V717I(Lon)
Hu V->F TTGTCATAGCAACCGTGATTTTCATCACCCTGGTGATGTTGAAG Indiana allele
Mouse B6J TTGTCATAGCAACCGTGATTGTCATCACCCTGGTGATGTTGAAG
A T V I V I T L V M L K
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
45231 | Fluorophore-1 | AAC CGT GAT TTT CAT CAC C | Quencher-1 | MUT Probe | ||
45232 | CGC CAT CAT CGG ACT CAT | Transgene Forward | A | |||
45233 | TGG ATG GAT GTG TAC TGT TTC TTC | Transgene Reverse | A | |||
oIMR1544 | CAC GTG GGC TCC AGC ATT | Internal Positive Control Forward | A | |||
oIMR3580 | TCA CCA GTC ATT TCT GCC TTT G | Internal Positive Control Reverse | A | |||
TmoIMR0105 | Fluorophore-2 | CCA ATG GTC GGG CAC TGC TCA A | Quencher-2 | IC Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
45232 | 0.40 uM |
45233 | 0.40 uM |
oIMR1544 | 0.40 uM |
oIMR3580 | 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 |