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Stock No: 001192
Protocol 348: Restriction Enzyme Digest Assay - Lepr<db>
Version 1.1
Notes
Publications are available which describe other approaches to genotyping this strain. One of these can be found at: https://www.nature.com/articles/aps201752
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 = 108 bp and 27 bp
Heterozygote = 27 bp, 108 bp and 135 bp
Wild type = 135 bp
Male mice have an unknown artifact band of ~ 170bp.
Add DNA Loading Dye** to digested products and separate on a 3% agarose gel.***
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| oIMR0985 | AGA ACG GAC ACT CTT TGA AGT CTC | Forward | ||||
| oIMR0986 | CAT TCA AAC CAT AGT TTA GGT TTG TGT | Reverse |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| AB PCR BufferII | 1.00 X |
| MgCl2 | 2.00 mM |
| dNTP | 0.20 mM |
| oIMR0985 | 1.00 uM |
| oIMR0986 | 1.00 uM |
| Taq DNA Polymerase | 0.03 U/ul |
| DNA |
Cycling
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 94.0 | -- | |
| 2 | 94.0 | -- | |
| 3 | 52.0 | -- | |
| 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 | |
| RsaI |
Strains Using This Protocol
| Stock Number | Strain Name |
|---|---|
| 002048 | B6 x C57BLKS-Dock7m Leprdb Myo15sh2-J/J |
| 000697 | B6.BKS(D)-Leprdb/J |
| 000699 | B6.Cg-Dock7m Leprdb/+ +/J |
| 005089 | B6.Cg-Qkiqk-2J/GrsrJ |
| 000642 | BKS.Cg-Dock7m +/+ Leprdb/J |
| 000700 | BKS.Cg-Dock7m Leprdb/+ +/J |
| 008340 | BKS.Cg-Leprdb Nos3tm1Unc/RhrsJ |
| 001192 | BKS.Cg-meaJ Leprdb +/+ + Dock7m/J |
| 000707 | CBA.Cg-Dock7m Leprdb/+ +/J |
| 012666 | D2.BKS(D)-Leprdb/J |
| 006654 | FVB.BKS(D)-Leprdb/ChuaJ |
| 11 strains use this protocol | |