Stock No: 006907
Protocol 26790: Standard PCR Assay - Tg(APOC3)3707Bres
Version 2.2
Notes
This assay will NOT distinguish hemizygous from homozygous transgenic animals.
Internal Positive Control (IPC) band does not typically amplify when transgene is present.
Internal Positive Control (IPC) band does not typically amplify when transgene is present.
The genotyping protocol(s) presented here have been optimized for reagents and conditions used by The Jackson Laboratory (JAX).
To genotype animals, JAX recommends researchers validate the assay independently upon receipt of animals into their facility.
Reaction cycling temperature and times may require additional optimization based on the specific genotyping reagents used.
Expected Results
Transgene = 173 bp
Internal positive control = 197 bp
Separated by gel electrophoresis on a 3.0% agarose gel.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 20286 | TGT CTC TGA ACC CTG GGA AG | Internal Positive Control Forward | A | |||
| 20287 | GCA CCT CCT TGA ACG ACA TC | Internal Positive Control Reverse | A | |||
| oIMR6799 | AGC TGG CAT AGC AGA GGT GT | Transgene Forward | A | |||
| oIMR6800 | GCA GCC TCT CAT TTG GAA AG | Transgene Reverse | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| 20286 | 0.50 uM |
| 20287 | 0.50 uM |
| oIMR6799 | 0.50 uM |
| oIMR6800 | 0.50 uM |
| Glycerol | 6.50 % |
| Dye | 1.00 X |
| Kapa 2G HS taq polym | 0.03 U/ul |
| DNA |
Cycling
| 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 |
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.
JAX uses a 'touchdown' cycling protocol and therefore has not calculated the optimal annealing temperature for each set of primers.
Strains Using This Protocol
This is the only strain that uses this protocol.