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Stock No: 006564
Protocol 764: Restriction Enzyme Digest Assay - Gusb<mps>
Version 1.1
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 = 95 bp
Heterozygote = 18 bp, 77 bp and 95 bp
Wild type = 77 bp and 18 bp
Heterozygote = 18 bp, 77 bp and 95 bp
Wild type = 77 bp and 18 bp
Separate by gel electrophoresis on a 5.0% agarose gel.**
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| oIMR6214 | CCT GTC TCA TTT GCA TGT G | |||||
| oIMR6215 | GAT AAC ATC CAC GTA CCG G |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| AB PCR BufferII | 1.00 X |
| MgCl2 | 2.00 mM |
| dNTP | 0.20 mM |
| oIMR6214 | 1.00 uM |
| oIMR6215 | 1.00 uM |
| DNA Loading Dye | 0.69 mM |
| Taq DNA Polymerase | 0.03 U/ul |
| DNA |
Cycling
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 94.0 | -- | |
| 2 | 94.0 | -- | |
| 3 | 55.0 | -- | |
| 4 | 72.0 | -- | repeat steps 2-4 for 35 cycles |
| 5 | 72.0 | -- | |
| 6 | 10.0 | -- | hold** |
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.
Reaction B
| Component | Final Concentration |
|---|---|
| AB PCR BufferII | X |
| ddH2O | |
| BSA | mg/ml |
| NciI |
Strains Using This Protocol
| Stock Number | Strain Name |
|---|---|
| 006564 | B6(C)-KitW-41J Gusbmps/BrkJ |
| 006559 | B6.C-H2-Kbm1/ByBir-Gusbmps/BrkJ |
| 000256 | B6.C-H2-Kbm1/ByBir-Gusbmps/J |
| 006562 | B6.CBy(Cg)-Gusbmps Gpi1a-m1J/BrkJ |
| 002086 | B6.Cg-Gusbmps Tg(Gussx)1Wat/J |
| 006407 | B6.Cg-Gusbmps/BrkJ |
| 005053 | NOD.Cg-Prkdcscid Gusbmps/SndsJ |
| 001880 | STOCK Gusbmps Tg(GUSB)4Sly/BirJ |
| 8 strains use this protocol | |