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Stock No: 006393
Protocol 22565: Standard PCR Assay - Adgrl1<tm1Sud>
Version 1.3
Notes
Re-named assay from Lphn1_tm1Sud to Adgrl1_tm1Sud to match allele name change. These primers are not ideal, if we are seeing issues with the assay re-designing with more optimized primer may be necessary. LM 12-26-19
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 = 450 bp
Heterozygote = 450 bp and 550 bp
Wild type = 550 bp
Heterozygote = 450 bp and 550 bp
Wild type = 550 bp
Separated by gel electrophoresis on a 1.5% agarose gel.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| oIMR6338 | CCC AAA AGA CAC TGT CTC CAA G | Common | A | |||
| oIMR6339 | CCG GCG CAT CAA CCC AAA TGG GAG CCC | Wild type | A | |||
| oIMR8574 | GAG CGC GCG CGG CGG AGT TGT TGA C | Mutant | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| oIMR6338 | 0.50 uM |
| oIMR6339 | 0.50 uM |
| oIMR8574 | 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.