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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 = ~245 bp
Heterozygote = ~155 bp and ~245 bp
Wild type = ~155 bp
Heterozygote = ~155 bp and ~245 bp
Wild type = ~155 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 |
|---|---|---|---|---|---|---|
| oIMR0180 | GCC TAG CCG AGG GAG AGC CG | Common | A | |||
| oIMR0181 | TGT GAC TTG GGA GCT CTG CAG C | Wild type Reverse | A | |||
| oIMR0182 | GCC GCC CCG ACT GCA TCT | 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 |
| oIMR0180 | 0.50 uM |
| oIMR0181 | 0.50 uM |
| oIMR0182 | 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 |
|---|---|
| 002246 | B6.129-Apoetm1Unc Ldlrtm1Her/J |
| 002052 | B6.129P2-Apoetm1Unc/J |
| 004631 | B6.Cg-Apoetm1Unc Cdh18Tg(GFAP-APOE_i4)1Hol/J |
| 007224 | B6.Cg-Apoetm1Unc (D12Mit182-D12Mit2)/Pgn |
| 008525 | B6.Cg-Tg(Cyp21a1-Apoe)619Fet/J |
| 004632 | B6.Cg-Tg(GFAP-APOE_i2)14Hol Apoetm1Unc/J |
| 004633 | B6.Cg-Tg(GFAP-APOE_i3)37Hol Apoetm1Unc/J |
| 002878 | B6;129-Apobtm1Sgy Apoetm1Unc/J |
| 002879 | B6;129-Apobtm2Sgy Apoetm1Unc/J |
| 002245 | B6;129-Apoetm1Unc Ldlrtm1Her/J |
| 004362 | B6;129-Scarb1tm1Kri Apoetm1Unc/J |
| 007067 | D2.129P2(B6)-Apoetm1Unc/J |
| 029818 | STOCK Apoetm1Unc Tg(APOE3*R224Q)1Her/J |
| 007069 | STOCK Apoetm1Unc/J |
| 14 strains use this protocol | |