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Stock No: 004597
Protocol 23492: Standard PCR Assay - Pten<tm1Hwu>
Version 3.3
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 = 328 bp
Heterozygote = 156 bp and 328 bp
Wild type = 156 bp
Heterozygote = 156 bp and 328 bp
Wild type = 156 bp
Separated by gel electrophoresis on a 3% agarose gel.
Sequence
Mutant PCR product sequence:
CAAGCACTCTGCGAACTGAGCTACATCCCAGAGTTCATACCAGGATTTAAGGATCTCAATAGGATAGAATCAAAACAGATACTAGTAAGATAAAAACCAGTAGTGATAGAACGGAAGTCTTGCTTCTAGTGCGGCCGCACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTTCAAGAATTCCGATCATATTCAATAACCCTTAATATAACTTCGTATAATGTATGCTATACGAAGTTATTAGGTCCCTCGAAGAGGTTCACTAGATAATAGCATCTTGCCTTCAAAAACTT
Yellow highlight: primer;
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| oIMR9554 | CAA GCA CTC TGC GAA CTG AG | Forward | A | |||
| oIMR9555 | AAG TTT TTG AAG GCA AGA TGC | Reverse | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTPS-kapa | 0.26 mM |
| oIMR9554 | 0.50 uM |
| oIMR9555 | 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.