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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 = ~179 bp
Heterozygote = ~179 bp and 351 bp
Wild type = 351 bp
Mut is GC rich
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 19799 | TAT GCA TCC CCA GTC TTT GG | Common | A | |||
| 19800 | CTA CCC AAC CAG CCC CTT AC | Wild type Reverse | A | |||
| oIMR7770 | ATA GAT TCG CCC TTG TGT CC | Mutant Reverse | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| 19799 | 0.50 uM |
| 19800 | 0.50 uM |
| oIMR7770 | 0.50 uM |
| Glycerol | 6.50 % |
| Dye | 1.00 X |
| Kapa 2G HS taq polym | 0.03 U/ul |
| DNA |
Cycling
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 94.0 | -- | |
| 2 | 94.0 | -- | |
| 3 | 65.0 | -- | -0.5 C per cycle decrease |
| 4 | 68.0 | -- | |
| 5 | -- | repeat steps 2-4 for 10 cycles (Touchdown) | |
| 6 | 94.0 | -- | |
| 7 | 60.0 | -- | |
| 8 | 72.0 | -- | |
| 9 | -- | repeat steps 6-8 for 28 cycles | |
| 10 | 72.0 | -- | |
| 11 | 10.0 | -- | hold |
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.
JAX uses a 'touchdown' cycling protocol and therefore has not calculated the optimal annealing temperature for each set of primers.
Strains Using This Protocol
| Stock Number | Strain Name |
|---|---|
| 007070 | AK.129S7(B6)-Ldlrtm1Her/J |
| 006952 | B6.129-Akt2tm1.1Mbb Ldlrtm1Her/J |
| 002246 | B6.129-Apoetm1Unc Ldlrtm1Her/J |
| 006883 | B6.129S7-Ldlrtm1Her Sod2tm1Leb/J |
| 002207 | B6.129S7-Ldlrtm1Her/J |
| 006580 | B6.Cg-Ins2Akita Ldlrtm1Her/J |
| 022488 | B6.Cg-Ldlrtm1Her Tg(Tagln-Pth1r*)2Towl/J |
| 006877 | B6.Cg-Ldlrtm1Her Tg(H2-K-AKR1B1)1Tj/J |
| 006906 | B6.Cg-Lepob Ldlrtm1Her/J |
| 002245 | B6;129-Apoetm1Unc Ldlrtm1Her/J |
| 003000 | B6;129S-Ldlrtm1Her Apobtm2Sgy/J |
| 002465 | B6;129S-Lrpap1tm1Her Ldlrtm1Her/J |
| 002077 | B6;129S7-Ldlrtm1Her/J |
| 012845 | CByJ.129S7(B6)-Ldlrtm1Her/J |
| 007068 | D2.129S7(B6)-Ldlrtm1Her/J |
| 004192 | STOCK Mttptm2Sgy Ldlrtm1Her Apobtm2Sgy Tg(Mx1-cre)1Cgn/J |
| 16 strains use this protocol | |