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This assay will NOT distinguish hemizygous from homozygous transgenic animals.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
oIMR3847 | GGT GAT GTC CAG CTT GGA GT | Transgene | A | |||
oIMR4110 | CCC CGT AAT GCA GAA GAA GA | Transgene | A | |||
oIMR7338 | CTA GGC CAC AGA ATT GAA AGA TCT | Internal Positive Control Forward | A | |||
oIMR7339 | GTA GGT GGA AAT TCT AGC ATC ATC C | 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 |
oIMR3847 | 0.50 uM |
oIMR4110 | 0.50 uM |
oIMR7338 | 0.50 uM |
oIMR7339 | 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 |
Stock Number | Strain Name |
---|---|
006055 | B6.Cg-Tg(CAG-Bgeo,-DsRed*MST)1Nagy/J |
006051 | B6.Cg-Tg(CAG-DsRed*MST)1Nagy/J |
006679 | B6;129P2-Or8a1tm27Mom/MomJ |
037280 | C57BL/6-Tg(Irgm1-DsRed2)1Nci/Mmjax |
005328 | NOD/ShiLt-Tg(Cd4-DsRed)4Lt/J |
005441 | STOCK Tg(CAG-DsRed*MST)1Nagy/J |
6 strains use this protocol |