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Transgene = 152 bp
WT = 287 bp
>chr17:29435540+29435826 287bp TTTAGTTGCAAGGGTTTCTGG CCAGCAAGATGTAGGAGGATG
This assay is capable of distinguishing hemi from hom. Transgene insertion site is known to be on mouse Chr 17.
This assay is NOT able to be used for copy number evaluation. If this is required, it is suggested to type by qPCR.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
53806 | TTT AGT TGC AAG GGT TTC TGG | Common | A | mChr17 | ||
53807 | GGG CGG AAG GAT CAG GAC | Mutant Reverse | A | Human UBC | ||
53886 | CCA GCA AGA TGT AGG AGG ATG | Wild type Reverse | A | mChr17 |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTP KAPA | 0.26 mM |
53806 | 0.50 uM |
53807 | 0.50 uM |
53886 | 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 |