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Technical Information Scientists
This assay does not work well without the use of a Hotstart Taq polymerase.
(a) We want to keep this as a generic statement, so that it covers the multiple
Hotstart Taqs we currently use, and if we change vendors in the future.
This assay will not distinquish hemizygous from homozygous transgenic animals.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
10012 | CCC AGG TTA CCA TGG AGA GA | Transgene Forward | 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 | |||
oIMR9402 | GAA CTT CAG GGT CAG CTT GC | Transgene Reverse | A |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTP KAPA | 0.26 mM |
10012 | 0.50 uM |
oIMR8744 | 0.50 uM |
oIMR8745 | 0.50 uM |
oIMR9402 | 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 |