Stock No: 002642
Protocol 28529: Standard PCR Assay - Gfap<tm1Mes>
Version 5.2
Notes
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
Mutant = 153bp
Heterozygote = 153bp and 420bp
Wild type = 420bp
Heterozygote = 153bp and 420bp
Wild type = 420bp
Separated by gel electrophoresis on a 1.5% agarose gel.<br />
<br />
If wt band in hets is weak w/AB Taq, no Abody@64C x30s suggest 45-60s or use Platinum Taq 64C x30s.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| oIMR0356 | GAT GGA GCG GAG ACG CAT CAC C | A | ||||
| oIMR0357 | TTG TCC CTC TCC ACC TCC AGC C | A | ||||
| oIMR1786 | GGA AGA CAA TAG CAG GCA TGC TGG | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| oIMR0356 | 0.50 uM |
| oIMR0357 | 0.50 uM |
| oIMR1786 | 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.