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This assay will NOT distinguish hemizygous from homozygous transgenic animals.
Transgene = ~415 bp
Internal positive control = 324 bp
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
15731 | CGG TGG TGC AGA TGA ACT T | Transgene Reverse | A | EYFP | ||
16072 | ACA GAC ACA CAC CCA GGA CA | Transgene Forward | A | Thy1 | ||
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 |
15731 | 0.50 uM |
16072 | 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 |
---|---|
007919 | B6.Cg-Tg(Thy1-EGFP)OJrs/GfngJ |
003782 | B6.Cg-Tg(Thy1-YFP)HJrs/J |
005627 | B6.Cg-Tg(Thy1-YFP/Syp)10Jrs/J |
011070 | B6;CBA-Tg(Thy1-EGFP)SJrs/NdivJ |
014130 | B6;CBA-Tg(Thy1-YFP)GJrs/GfngJ |
025018 | D2.Cg-Gpnmb+Tg(Thy1-YFP)HJrs/SjJ |
024705 | D2.Cg-Tg(Thy1-YFP)HJrs/SjJ |
025019 | D2.Cg-Tg(Thy1-YFP/Syp)10Jrs/SjJ |
007788 | STOCK Tg(Thy1-EGFP)MJrs/J |
9 strains use this protocol |