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Stock No: 003968
Protocol 23475: Standard PCR Assay - Il10<tm1Cgn>
Version 2.3
Notes
Use Perfect Match© (Stratagene) added before Taq to delete non-specific bands.
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 = 312 bp
Heterozygote = 137 bp and 312 bp
Wild type = 137 bp
Heterozygote = 137 bp and 312 bp
Wild type = 137 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 |
|---|---|---|---|---|---|---|
| oIMR7376 | CCA CAC GCG TCA CCT TAA TA | Mutant Reverse | A | |||
| oIMR9573 | CTT GCA CTA CCA AAG CCA CA | Common | A | |||
| oIMR9574 | GTT ATT GTC TTC CCG GCT GT | 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 |
| oIMR7376 | 0.50 uM |
| oIMR9573 | 0.50 uM |
| oIMR9574 | 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
| Stock Number | Strain Name |
|---|---|
| 004368 | 129(B6)-Il10tm1Cgn/J |
| 002250 | B10.129P2(B6)-Il10tm1Cgn/J |
| 002251 | B6.129P2-Il10tm1Cgn/J |
| 005912 | B6.Cg-Il10tm1Cgn (D3Mit49-D3Mit348)/Lt |
| 004333 | C.129P2(B6)-Il10tm1Cgn/J |
| 003968 | C3Bir.129P2(B6)-Il10tm1Cgn/LtJ |
| 036145 | CBA.129P2(B6)-Il10tm1Cgn/TrnrJ |
| 004291 | NOD.129P2(Cg)-Il10tm1Cgn Il4tm1Cgn/DvsJ |
| 004266 | NOD.129P2(Cg)-Il10tm1Cgn/DvsJ |
| 005346 | NOD.Cg-Il10tm1Cgn Casp1tm1Sesh Casp4del/LtJ |
| 004228 | NOD.Cg-Il10tm1Cgn Il4tm1Cgn/Dvs |
| 11 strains use this protocol | |