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This assay will NOT distinguish hemizygous from homozygous transgenic animals.
Have put internal controls on all 3 protocols. But only 1 set needs to be run if ithey are run separated.
Transgene = 671 bp
Internal Control = 324 bp
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
12274 | CTG GAG TTG TCC CAA TTC TTG TTG AAT TAG | Transgene Forward | ||||
12275 | GCC ATG TGT AAT CCC AGC AGC TGT TAC | Transgene Reverse | ||||
oIMR7338 | CTA GGC CAC AGA ATT GAA AGA TCT | |||||
oIMR7339 | GTA GGT GGA AAT TCT AGC ATC ATC C |
Component | Final Concentration |
---|---|
ddH2O | |
KAPA Rxn Buffer Contains 1.5 mM Mg2+ final concentration. | 1.00 |
dNTP | 0.20 |
12274 | 1.00 |
12275 | 1.00 |
DNA Loading Dye | 0.69 |
KAPATaq DNA Polymera No proofreading function. 1 error per 40000 nucleotides. Initial denature 2 min. | 0.03 |
DNA | 0.00 |
Step | Temp °C | Time | Note |
---|---|---|---|
1 | 94.0 | -- | |
2 | 94.0 | -- | |
3 | 64.0 | -- | |
4 | 72.0 | -- | |
5 | -- | Go to step 2-4, 35 times | |
6 | 72.0 | -- | |
7 | 10.0 | -- | hold |
Component | Final Concentration |
---|---|
ddH2O | |
KAPA Rxn Buffer Contains 1.5 mM Mg2+ final concentration. | 1.00 |
dNTP | 0.20 |
oIMR7338 | 0.75 |
oIMR7339 | 0.75 |
DNA Loading Dye | 0.69 |
KAPATaq DNA Polymera No proofreading function. 1 error per 40000 nucleotides. Initial denature 2 min. | 0.03 |
DNA |
Step | Temp °C | Time | Note |
---|---|---|---|
1 | 94.0 | -- | |
2 | 94.0 | -- | |
3 | 64.0 | -- | |
4 | 72.0 | -- | |
5 | -- | step 2-4,35 cycles | |
6 | 72.0 | -- | |
7 | 10.0 | -- | Forever |