For in-depth product & services help, ask our
Technical Information Scientists
Stock No: 002878
Protocol 315: Restriction Enzyme Digest Assay - Apob<tm1Sgy>
Version 2.1
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 = 181 bp and 100 bp
Heterozygote = 100 bp, 181 bp and 281 bp
Wild type = 281 bp
Heterozygote = 100 bp, 181 bp and 281 bp
Wild type = 281 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 |
|---|---|---|---|---|---|---|
| oIMR0671 | CTG AAT GCA TCT GAC TGG GAG AGA | Common | ||||
| oIMR0672 | CGG ATA TGA TAC TGT TCA TCA AGA A | Common |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| AB PCR BufferII | 1.00 X |
| MgCl2 | 1.50 mM |
| dNTP | 0.20 mM |
| oIMR0671 | 1.00 uM |
| oIMR0672 | 1.00 uM |
| DNA Loading Dye | 0.69 mM |
| Taq DNA Polymerase | 0.01 U/ul |
| DNA |
Cycling
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 94.0 | -- | |
| 2 | 94.0 | -- | |
| 3 | 64.0 | -- | *-0.5 degrees per cycle |
| 4 | 72.0 | -- | repeat steps 2-4 for 12 cycles |
| 5 | 94.0 | -- | |
| 6 | 58.0 | -- | |
| 7 | 72.0 | -- | repeat steps 5-7 for 25 cycles |
| 8 | 72.0 | -- | |
| 9 | 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 |
| Spe I |