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Notes
This assay does not work well without the use of a Hotstart (We are using Taq Start Antibody mixed 1:1 with Taq polymerase).
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
Transgene = 166 bp
Internal positive control = 324 bp
Internal positive control = 324 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 |
|---|---|---|---|---|---|---|
| oIMR1241 | ATG ACC GAG TAC AAG CCC AC | Mutant Forward | A | Puro F | ||
| oIMR1242 | GCG TGA GGA AGA GTT CTT GC | Mutant Reverse | A | Puro R | ||
| oIMR7338 | CTA GGC CAC AGA ATT GAA AGA TCT | Internal Positive Control Forward | A | |||
| oIMR7339 | GTA GGT GGA AAT TCT AGC ATC ATC C | Internal Positive Control Reverse | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| oIMR1241 | 0.50 uM |
| oIMR1242 | 0.50 uM |
| oIMR7338 | 0.50 uM |
| oIMR7339 | 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 |
|---|---|
| 024906 | 129-Dp(16Tiam1-Il10rb)8Yey/J |
| 039525 | B6.129(Cg)-Shank3em1Yhj/J |
| 040296 | B6.Cg-Gt(ROSA)26Sorem2(CAG-LOC4337827*)Mbu Ikzf1em1Mbu Ikzf3em1Mbu/J |
| 040297 | B6.Cg-Gt(ROSA)26Sorem2(CAG-LOC4337827*)Mbu Ikzf1em1Mbu/J |
| 040298 | B6.Cg-Gt(ROSA)26Sorem2(CAG-LOC4337827*)Mbu Ikzf3em1Mbu/J |
| 040295 | B6.Cg-Gt(ROSA)26Sorem2(CAG-LOC4337827*)Mbu Tcf3em1Mbu/J |
| 040300 | B6.Cg-Gt(ROSA)26Sorem2(CAG-LOC4337827*)Mbu Tcf4em1Mbu/J |
| 035506 | B6.Cg-Gt(ROSA)26Sortm3(CEBPB)Myz/J |
| 040293 | B6.Cg-Pax5em1Mbu Gt(ROSA)26Sorem2(CAG-LOC4337827*)Mbu/J |
| 014168 | B6;129-Pot1atm1.1Tdl/J |
| 038621 | B6;129P2-Ctcftm2.2Gal/J |
| 039661 | B6;129P2-Gt(ROSA)26Sorem2(CAG-LOC4337827*)Mbu/J |
| 002355 | C57BL/6J-Tg(pPWL88puro)2Ems/J |
| 003208 | STOCK Dnmt1tm3Jae Hprt1b-m3 Tg(pPWL512hyg)1Ems/J |
| 040294 | STOCK Gt(ROSA)26Sorem2(CAG-LOC4337827*)Mbu Ebf1em1Mbu/J |
| 040299 | STOCK Gt(ROSA)26Sorem2(CAG-LOC4337827*)Mbu Prdm1em2Mbu/J |
| 018121 | STOCK Gt(ROSA)26Sortm1(birA)Mejr Gata4tm3.1Wtp/J |
| 028980 | STOCK Ppm1dtm1Lad/J |
| 18 strains use this protocol | |