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Stock No: 012709
Protocol 28387: Standard PCR Assay - Il4ra<tm3.1Tch>
Version 2.2
Notes
This assay does not work well without the use of a Hotstart Taq polymerase.
When using the two primers flanking the loxp sites (10858/10859), the assay may not reliably amplify and separate both fragments from heterozygous mutant mice. Therefore we have included a mutant specific (loxp) primer that can be used to amplify from the mutant allele (oIMR9042).
When using the two primers flanking the loxp sites (10858/10859), the assay may not reliably amplify and separate both fragments from heterozygous mutant mice. Therefore we have included a mutant specific (loxp) primer that can be used to amplify from the mutant allele (oIMR9042).
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
Primers 10858/10859:
Mutant =479 bp
Heterozygote =479bp, and 463 bp
Wild type = 463bp
Primers oIMR9042/10859:
Mutant = ~ 185 bp
Wild type = no amplification
Mutant =479 bp
Heterozygote =479bp, and 463 bp
Wild type = 463bp
Primers oIMR9042/10859:
Mutant = ~ 185 bp
Wild type = no amplification
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 10858 | GGC TGT CTA TTT TAG GTG CC | A | ||||
| 10859 | TCT TCT TCT TTA CTC TGT GCT | A | ||||
| oIMR9042 | CTT CGT ATA GCA TAC ATT ATA CG | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| 10858 | 0.50 uM |
| 10859 | 0.50 uM |
| oIMR9042 | 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.