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This assay will NOT distinguish hemizygous from homozygous transgenic animals.
This assay does not work well without use of Hotstart Taq polymerase.
The transgene primers used in this assay are identical to those used to type JR015814- VAMP assay. These detect the Vamp2 cDNA sequence of the transgene.
Transgene = ~300bp (Tg primers can amplify a ~900bp wildtype product)
Internal Positive Control = 200bp
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
11857 | CTG CAC CTC CTC CAA ACC TTA CTA | Transgene Forward | A | |||
11858 | ATG ATG AGG ATG ATG GCG CAG A | Transgene Reverse | A | |||
oIMR8744 | CAA ATG TTG CTT GTC TGG TG | Internal Positive Control Forward | A | |||
oIMR8745 | GTC AGT CGA GTG CAC AGT TT | Internal Positive Control Reverse | A |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTP KAPA | 0.26 mM |
11857 | 0.50 uM |
11858 | 0.50 uM |
oIMR8744 | 0.50 uM |
oIMR8745 | 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 |