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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 = ~280 bp
Heterozygote = ~280 bp and 200 bp
Wild type = 200 bp
Sequence
AAGTGTCACTCTGACGGGTTCTTCTGGAGGAGATCCTTCTTTTGTTATCAAGCTTATCGCGCGTGTGGCCTCGAACACCGAGCGACCCTCGACCATGCCCAAGAAGAAGAGGAAGGTGTCCAATTTACTGACCGTACACCAAAATTTGCCTGCATTACCGGTCGATGCAACGAGTGATGAGGTTCGCAAGAACCTGATGGACATGTTCAGGGATCGCCAGGCGTTTTCTGAGCATACCTGGAAAATG
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 25540 | CAC ACT GCT CTT CCA AGT GTC | Common | A | |||
| 25541 | AGT TAC CTT AAT AGC AGA CAG ATC G | Wild type Reverse | A | |||
| oIMR1709 | GCA AAC GGA CAG AAG CAT TT | Mutant Reverse | A | Cre R |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 0.24 X |
| MgCl2 | 0.48 mM |
| dNTP KAPA | 0.05 mM |
| 25540 | 0.12 U |
| 25541 | 0.12 U |
| oIMR1709 | 0.12 U |
| Kapa 2G HS taq polym | 0.00 U/ul |
| EvaGreen | 0.24 X |
| 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.