Stock No: 002877
Protocol 550: Restriction Enzyme Digest Assay - Apob<tm2Sgy>
Version 1.1
Notes
This assay does not work well without the use of a Hotstart (We are using Taq Start Antibody mixed 1:1 with Taq polymerase).
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 = 2500 bp and 300 bp
Heterozygotes = 2800 bp and 2500 bp and 300 bp
Wild type = 2800 bp (uncut)
Heterozygotes = 2800 bp and 2500 bp and 300 bp
Wild type = 2800 bp (uncut)
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 |
|---|---|---|---|---|---|---|
| oIMR1745 | TCC TGA GCT GCT GTA GTT TTT C | Common | ||||
| oIMR1776 | CCG CAT GAA GAC TGA CTC TG | Common |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| AB PCR BufferII | 1.00 X |
| MgCl2 | 2.50 mM |
| dNTP | 0.20 mM |
| oIMR1776 | 0.50 uM |
| oIMR1745 | 0.50 uM |
| Taq DNA Polymerase + | 0.02 U/ul |
| DNA |
Cycling
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 94.0 | -- | |
| 2 | 94.0 | -- | |
| 3 | 65.0 | -- | |
| 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 |
| Hind III |