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This assay does not work well without the use of a HotstartTaq polymerase. This assay cannot distinguish heterozygous from homozygous KO mice.
Mutant = ~200 bp
Heterozygote = ~200 bp and 487 bp
Wild type = 487 bp
A duplication of part of the Aim2 genomic region at the 3' end of the integration site makes this assay unable to distinguish heterozygous from homozygous KO mice by PCR.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
12906 | GTT TGG CTC AGA AAT GTC CAG | Common | A | |||
12907 | CCC ACA TAA CCT GGG ATT AGT T | Wild type Reverse | A | |||
12908 | AAG GGT CTT TGA GCA CCA GA | Mutant Reverse | A |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTP KAPA | 0.26 mM |
12906 | 0.50 uM |
12907 | 0.50 uM |
12908 | 0.50 uM |
Glycerol | 6.50 % |
Dye | 1.00 X |
Kapa 2G HS taq polym | 0.03 U/ul |
DNA |
Step | Temp °C | Time | Note |
---|---|---|---|
1 | 94.0 | -- | |
2 | 94.0 | -- | |
3 | 65.0 | -- | -0.5 C per cycle decrease |
4 | 68.0 | -- | |
5 | -- | repeat steps 2-4 for 10 cycles (Touchdown) | |
6 | 94.0 | -- | |
7 | 60.0 | -- | |
8 | 72.0 | -- | |
9 | -- | repeat steps 6-8 for 28 cycles | |
10 | 72.0 | -- | |
11 | 10.0 | -- | hold |