For in-depth product & services help, ask our
Technical Information Scientists
Stock No: 006867
Protocol 176: Restriction Enzyme Digest Assay - Ins2<Akita>
Version 1.1
Notes
Incubate 8 µl restriction digest with 4 µl PCR product for a minimum of 3 hours at 37 C prior to running on gel.
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 = 280 bp
Heterozygote = 140 bp and 280 bp
Wild type = 140 bp
Heterozygote = 140 bp and 280 bp
Wild type = 140 bp
Separate digested products on a 3.0% agarose gel.**
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| oIMR1093 | TGC TGA TGC CCT GGC CTG CT | Forward | ||||
| oIMR1094 | TGG TCC CAC ATA TGC ACA TG | Reverse |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| AB PCR BufferII | 1.00 X |
| MgCl2 | 2.00 mM |
| dNTP | 0.20 mM |
| oIMR1093 | 0.80 uM |
| oIMR1094 | 0.80 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 C 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 |
| Fnu4HI |
Strains Using This Protocol
| Stock Number | Strain Name |
|---|---|
| 012307 | B6.129-Apoetm3(APOE*4)Mae Ldlrtm1(LDLR)Mae/J |
| 006860 | B6.Cg-Ins2Akita Bdkrb2tm1Jfh/SmiJ |
| 006580 | B6.Cg-Ins2Akita Ldlrtm1Her/J |
| 004369 | B6.Cg-Rag1tm1Mom Ins2Akita/J |
| 003548 | C57BL/6-Ins2Akita/J |
| 007562 | D2.B6-Ins2Akita/MatbJ |
| 006867 | FVB.B6-Ins2Akita/MlnJ |
| 008659 | NOD.Cg-Rag1tm1Mom Ins2Akita Prf1tm1Sdz/SzJ |
| 014568 | NOD.Cg-Rag1tm1Mom Ins2Akita Il2rgtm1Wjl/SzJ |
| 9 strains use this protocol | |