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Stock No: 035973
Protocol 40420: Standard PCR Assay - Rpe65<tm1.1(cre/ERT2)Kser>
Version 1.0
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
>chr3:159624750+159624952 203bp TGGAGACTAATATCCCTGTGACC TGTGTTCTGCAATGTGTGTGT
Mutant = ~300 bp
Heterozygote = ~300 bp and 203 bp
Wild type = 203 bp
Sequence
MUT SEQ:
TCTGAGTGTGGTGGTGAGCCCTGGGGCAGGGCAAAAGCCTGCATATCTCCTGGTTCTGAATGCCAAAGACTTGAGTGAAATTGCCAGGGCTGAAGTGGAGACTAATATCCCTGTGACCTTCCATGGACTGTTCAAAAGATCCACGCGTGGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCTATGTCCAATTTACTGACCGTACACCAAAATTTGCCTGCATTACCGGTCGATGCAACGAGTGATGAGGTTCGCAAGAACCTGATGGACATGTTCAGGGATCGCCAGGCGTTTTCTGAGCATACCTGGAAAATGCTTCTGTCCGTTTGCCGGTCGTGGGCGGCATGGTGCAAG
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 14314 | CGG TTA TTC AAC TTG CAC CA | Mutant Reverse | A | |||
| 55994 | TGG AGA CTA ATA TCC CTG TGA CC | Common | A | |||
| 55995 | TGT GTT CTG CAA TGT GTG TGT | Wild type Reverse | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| 14314 | 0.50 uM |
| 55994 | 0.50 uM |
| 55995 | 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.