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Stock No: 002171
Protocol 11947: Standard PCR Assay - Hprt1<b-m3>
Version 3.0
Notes
This assay does work well without the use of a Hotstart Taq polymerase.
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 =~ 300 bp
Heterozygote =~300 bp and 402 bp
Wild type = 402 bp
Heterozygote =~300 bp and 402 bp
Wild type = 402 bp
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 11428 | TAC ATG CAG GCA CTC ACA CA | Common | ||||
| 11429 | CAT GGA ATC CAG AGA AGG ACA | Wild type Forward | ||||
| 11430 | ATT CCT GCC CCA ACA ATG AT | Mutant Forward |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.00 |
| MgCl2 | 2.00 |
| dNTP KAPA | 0.20 |
| Primer 1 | 0.50 |
| Primer 2 | 0.50 |
| Primer 3 | 0.50 |
| 10x Loading Dye | 0.50 |
| Kapa 2G HS taq polym | 0.01 |
| 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.