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Stock No: 008080
Protocol 23355: Standard PCR Assay - TG(CAG-SAC/EGFP)
Version 2.3
Notes
This assay does not work well without the use of a Hotstart (We are using Taq Start Antibody mixed 1:1 with Taq polymerase).
Be aware: At lower annealing temps, an endogenous band of ~285bp can be amplified. A 3.0% gel can distinguish this band from the transgene band.
This assay will NOT distinguish hemizygous from homozygous transgenic animals.
Be aware: At lower annealing temps, an endogenous band of ~285bp can be amplified. A 3.0% gel can distinguish this band from the transgene band.
This assay will NOT distinguish hemizygous from homozygous transgenic animals.
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 = 247 bp
Wild type = 324 bp
Wild type = 324 bp
Separated by gel electrophoresis on a 1.5% agarose gel.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 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 | |||
| oIMR8991 | CGA AGC AGG ACA GAA GGA AC | Forward | A | |||
| oIMR9402 | GAA CTT CAG GGT CAG CTT GC | Mutant Reverse | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| oIMR7338 | 0.50 uM |
| oIMR7339 | 0.50 uM |
| oIMR8991 | 0.50 uM |
| oIMR9402 | 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.