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Notes
Mut = GCG
WT= CGC
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
Sequence
SEQ (bp changes in brackets with wt first):
tcaccccttcccaaactacattcggccgcggacactgcagccgcccgcatcctcccgccgccgtcacttccatcacgcgttgccacctgcg(CGC/GCG)caccatccggatctggaggcccaggccaggcgcgcgcaggaggaagcggacgcggaggagcgccggctgcaggagcaggaggagctggagaattaca
Sequence notes have all bp changes noted within plus or minus 30 bp of mutation of interest. Mutation of interest in bold and red. “Nucleotide changes in bold and red are mutations of interest. Other bp changes (plus or minus X) are silent mutations.”
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 72816 | CCA GCA GAC GAT GTG GTC AG | Mutant Forward | A | |||
| 72817 | GCG TCC GCT TCC TCC T | Mutant Reverse | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTPS-kapa | 0.26 mM |
| 72816 | 0.50 uM |
| 72817 | 0.50 uM |
| Glycerol | 6.50 % |
| 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.