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Stock No: 015840
Protocol 8546: Standard PCR Assay - Itga8<tm1.1Rdav>
Version 2.0
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 = 278 bp
Heterozygote = 244 bp and 278 bp
Wild type = 244 bp
<p>Band sizes close on 1.5% gel. Could try 3.0% for better separation. </p>
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 12278 | TCC TTG TCT TCC TGT GTA TTT GAC | Forward | ||||
| 12279 | TGT CTG AGA AGA TTC AGC AGT GGG | Reverse |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.00 |
| MgCl2 | 2.00 |
| dNTP KAPA | 0.20 |
| 12278 | 0.50 |
| 12279 | 0.50 |
| 10x Loading Dye | 0.50 |
| Kapa 2G HS taq polym | 0.01 |
| EvaGreen | 1.00 |
| DNA |
Cycling
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 94.0 | -- | |
| 2 | 94.0 | -- | |
| 3 | 65.0 | -- | -1.5 C per cycle decrease |
| 4 | 68.0 | -- | |
| 5 | -- | repeat steps 2-4 for 10 cycles | |
| 6 | 94.0 | -- | |
| 7 | 50.0 | -- | |
| 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.
Strains Using This Protocol
This is the only strain that uses this protocol.